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J Pharm Sci. 2017 Aug;106(8):1961-1970. doi: 10.1016/j.xphs.2017.04.037. Epub 2017 Apr 26.

Optimization of the Production Process and Characterization of the Yeast-Expressed SARS-CoV Recombinant Receptor-Binding Domain (RBD219-N1), a SARS Vaccine Candidate.

Author information

1
Texas Children's Hospital Center for Vaccine Development, Houston, Texas 77030.
2
Texas Children's Hospital Center for Vaccine Development, Houston, Texas 77030; Department of Pediatrics, National School of Tropical Medicine, Baylor College of Medicine, Houston, Texas 77030; Department of Molecular Virology and Microbiology, National School of Tropical Medicine, Baylor College of Medicine, Houston, Texas 77030.
3
Texas Children's Hospital Center for Vaccine Development, Houston, Texas 77030; Department of Pediatrics, National School of Tropical Medicine, Baylor College of Medicine, Houston, Texas 77030; Department of Molecular Virology and Microbiology, National School of Tropical Medicine, Baylor College of Medicine, Houston, Texas 77030; Department of Biology, Baylor University, Waco, Texas 76798. Electronic address: bottazzi@bcm.edu.
4
Texas Children's Hospital Center for Vaccine Development, Houston, Texas 77030; Department of Pediatrics, National School of Tropical Medicine, Baylor College of Medicine, Houston, Texas 77030; Department of Molecular Virology and Microbiology, National School of Tropical Medicine, Baylor College of Medicine, Houston, Texas 77030; Department of Biology, Baylor University, Waco, Texas 76798; James A. Baker III Institute for Public Policy, Rice University, Houston, Texas 77005.

Abstract

From 2002 to 2003, a global pandemic of severe acute respiratory syndrome (SARS) spread to 5 continents and caused 8000 respiratory infections and 800 deaths. To ameliorate the effects of future outbreaks as well as to prepare for biodefense, a process for the production of a recombinant protein vaccine candidate is under development. Previously, we reported the 5 L scale expression and purification of a promising recombinant SARS vaccine candidate, RBD219-N1, the 218-amino acid residue receptor-binding domain (RBD) of SARS coronavirus expressed in yeast-Pichia pastoris X-33. When adjuvanted with aluminum hydroxide, this protein elicited high neutralizing antibody titers and high RBD-specific antibody titers. However, the yield of RBD219-N1 (60 mg RBD219-N1 per liter of fermentation supernatant; 60 mg/L FS) still required improvement to reach our target of >100 mg/L FS. In this study, we optimized the 10 L scale production process and increased the fermentation yield 6- to 7-fold to 400 mg/L FS with purification recovery >50%. A panel of characterization tests indicated that the process is reproducible and that the purified, tag-free RBD219-N1 protein has high purity and a well-defined structure and is therefore a suitable candidate for production under current Good Manufacturing Practice and future phase-1 clinical trials.

KEYWORDS:

Pichia pastoris; circular dichroism; hydrophobic interaction chromatography; protein characterization; protein purification

PMID:
28456726
PMCID:
PMC5612335
DOI:
10.1016/j.xphs.2017.04.037
[Indexed for MEDLINE]
Free PMC Article

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