Title
Optimization of the production process and characterization of the yeast-expressed SARS-CoV recombinant receptor-binding domain (RBD219-N1), a SARS vaccine candidate
Author
Wen-Hsiang Chen, Shivali M. Chag, Mohan V. Poongavanam, Amadeo B. Biter, Ebe A. Ewere, Wanderson Rezende, Christopher A. Seid, Elissa M. Hudspeth, Jeroen Pollet, C. Patrick McAtee, Ulrich Strych, Maria Elena Bottazzi, Peter J. Hotez
Year
2017
Journal
Journal of Pharmaceutical Sciences
Abstract
From 2002 to 2003, a global pandemic of severe acute respiratory syndrome (SARS) spread to five continents and caused 8,000 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 6-7-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 cGMP and future Phase 1 clinical trials.
Instrument
J-1500
Keywords
Circular dichroism, Secondary structure, Thermal stability, Thermodynamics, Biochemistry, Pharmaceutical