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

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

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.

MeSH terms

  • Cloning, Molecular / methods
  • Fermentation
  • Humans
  • Industrial Microbiology / methods
  • Pichia / genetics*
  • Protein Domains
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • SARS Virus / chemistry
  • SARS Virus / genetics*
  • Severe Acute Respiratory Syndrome / prevention & control
  • Severe Acute Respiratory Syndrome / virology
  • Spike Glycoprotein, Coronavirus / chemistry
  • Spike Glycoprotein, Coronavirus / genetics*
  • Spike Glycoprotein, Coronavirus / isolation & purification
  • Vaccines, Synthetic / chemistry
  • Vaccines, Synthetic / genetics*
  • Vaccines, Synthetic / isolation & purification
  • Viral Vaccines / chemistry
  • Viral Vaccines / genetics*
  • Viral Vaccines / isolation & purification

Substances

  • Recombinant Proteins
  • Spike Glycoprotein, Coronavirus
  • Vaccines, Synthetic
  • Viral Vaccines
  • spike glycoprotein, SARS-CoV