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

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Dux M., Barent R., Sinha J., GOUTHRO M., Swanson T., Barthuli A., ...Daha Fazla

PROTEIN EXPRESSION AND PURIFICATION, cilt.45, sa.2, ss.359-367, 2006 (SCI-Expanded) identifier identifier identifier

  • Yayın Türü: Makale / Tam Makale
  • Cilt numarası: 45 Sayı: 2
  • Basım Tarihi: 2006
  • Doi Numarası: 10.1016/j.pep.2005.08.015
  • Dergi Adı: PROTEIN EXPRESSION AND PURIFICATION
  • Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus
  • Sayfa Sayıları: ss.359-367
  • Anahtar Kelimeler: botulinum, Pichia pastoris, ion-exchange chromatography, hydrophobic charge induction chromatography, homogenized, filtration, recombinant protein, scale-lip, TOXIN, FERMENTATION, EFFICACY
  • Akdeniz Üniversitesi Adresli: Hayır

Özet

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.