Botulinum - Research - Purification and Scale-Up, Serotype E
Author:
Dux, MP, Barent, R, Sinha, J, Gouthro, M, Swanson, T, Barthuli, A, Inan, M, Ross, JT, Smith, LA, Smith, TJ, Webb, R, Loveless, B, Henderson, I and Meagher, MMSummary:
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 (IEC) steps (SP Sepharose Fast Flow, Q Sepharose Fast Flow, Sp Sepharose High Performance) and polished with a hydrophobic charge induction chromatography (HCIC) step (MEP HyperCel„§).
Method development at the bench scale was achieved using 7 ¡V 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 1.01 grams of rBoNTE(Hc) / kg cells at the bench scale and 580 mg 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 1,000 mouse intraperitoneal LD50¡¦s of BoNT/E.
Related Links
Check out our Animal Rule FAQs.
View more publications by topic in our Knowledge Library.