title: Cell bank characterization and fermentation optimization for production of recombinant heavy chain C-terminal fragment of botulinum neurotoxin serotype E (rBoNTE(H(c)): antigen E) by Pichia pastoris.
abstract: A process was developed for production of a candidate vaccine antigen, recombinant C-terminal heavy chain fragment of the botulinum neurotoxin serotype E, rBoNTE(H(c)) in Pichia pastoris. P. pastoris strain GS115 was transformed with the rBoNTE(H(c)) gene inserted into pHILD4 Escherichia coli-P. pastoris shuttle plasmid. The clone was characterized for genetic stability, copy number, and BoNTE(H(c)) sequence. Expression of rBoNTE(H(c)) from the Mut(+) HIS4 clone was confirmed in the shake-flask, prior to developing a fed-batch fermentation process at 5 and 19 L scale. The fermentation process consists of a glycerol growth phase in batch and fed-batch mode using a defined medium followed by a glycerol/methanol transition phase for adaptation to growth on methanol and a methanol induction phase resulting in the production of rBoNTE(H(c)). Specific growth rate, ratio of growth to induction phase, and time of induction were critical for optimal rBoNTE(H(c)) production and minimal proteolytic degradation. A computer-controlled exponential growth model was used for process automation and off-gas analysis was used for process monitoring. The optimized process had an induction time of 9 h on methanol and produced up to 3 mg of rBoNTE(H(c)) per gram wet cell mass as determined by HPLC and Western blot analysis.
authors.affiliation: Biological Process Development Facility, Department of Chemical and Biomolecular Engineering, University of Nebraska-Lincoln, Lincoln, NE 68588-0466, USA.
authors.forename: Jayanta
authors.initials: J
authors.lastname: Sinha
authors.forename: Mehmet
authors.initials: M
authors.lastname: Inan
authors.forename: Sarah
authors.initials: S
authors.lastname: Fanders
authors.forename: Shinichi
authors.initials: S
authors.lastname: Taoka
authors.forename: Mark
authors.initials: M
authors.lastname: Gouthro
authors.forename: Todd
authors.initials: T
authors.lastname: Swanson
authors.forename: Rick
authors.initials: R
authors.lastname: Barent
authors.forename: Ardis
authors.initials: A
authors.lastname: Barthuli
authors.forename: Bonnie M
authors.initials: BM
authors.lastname: Loveless
authors.forename: Leonard A
authors.initials: LA
authors.lastname: Smith
authors.forename: Theresa
authors.initials: T
authors.lastname: Smith
authors.forename: Ian
authors.initials: I
authors.lastname: Henderson
authors.forename: John
authors.initials: J
authors.lastname: Ross
authors.forename: Michael M
authors.initials: MM
authors.lastname: Meagher
mesh_terms: D001428:Bacterial Vaccines / Q000096:biosynthesis* / Q000235:genetics; D018070:Biological Specimen Banks / Q000592:standards; D019149:Bioreactors / Q000382:microbiology / Q000592:standards; D001905:Botulinum Toxins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D003198:Computer Simulation; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics / Q000302:isolation & purification; D007218:Industrial Microbiology / Q000592:standards; D008954:Models, Biological; D010843:Pichia / Q000166:cytology* / Q000235:genetics / Q000254:growth & development*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D001428:Bacterial Vaccines; D011994:Recombinant Proteins; D001905:Botulinum Toxins; C096326:botulinum toxin type E