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Purification and scale-up of a recombinant heavy chain fragment C of botulinum neurotoxin serotype E in Pichia pastoris GS115

  1. Author:
    Dux, M. P.
    Barent, R.
    Sinha, J.
    Gouthro, M.
    Swanson, T.
    Barthuli, A.
    Inan, M.
    Ross, J. T.
    Smith, L. A.
    Smith, T. J.
    Webb, R.
    Loveless, B.
    Henderson, I.
    Meagher, M. M.

  2. Author Address

    Univ Nebraska, Dept Chem Engn, Biol Proc Dev Facil, Lincoln, NE 68588 USA. A CSC Co, LLC, DVC, Frederick, MD 21702 USA. USA, Med Res Inst Infect Dis, Div Toxicol, Frederick, MD 21702 USA.;Meagher, MM, Univ Nebraska, Dept Chem Engn, Biol Proc Dev Facil, Lincoln, NE 68588 USA.;mmeagher1@unl.edu
    1. Year: 2006
    2. Date: Feb
  2. Journal: Protein Expression and Purification
    1. 45
    2. 2
    3. Pages: 359-367
  4. Type of Article: Article
  5. ISSN: 1046-5928
  1. 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(H-c), was produced intracellular in Pichict 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 7380 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 g of rBoNTE(H-c)/kg cells at the bench scale and 580 mg vaccine/kg cells at the pilot scale. N-terminal sequencing showed that the purified rBoNTE(H-c) N-terminus is intact and was found to protect mice against a challenge of 1000 mouse intraperitoneal LD50'S Of BoNT/E. (c) 2005 Elsevier Inc. All rights reserved.

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  2. DOI: 10.1016/j.pep.2005.08.015
  3. View record in Web of Science®
  4. WOS: 000235247700015

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