Optimization of the Production Process and Characterization of the Yeast-Expressed SARS-CoV Recombinant Receptor-Binding Domain (RBD219-N1), a SARS Vaccine Candidate
| Autor: | Jeroen Pollet, Wanderson C. Rezende, Shivali M. Chag, Elissa M. Hudspeth, Wen-Hsiang Chen, Peter J. Hotez, Maria Elena Bottazzi, Mohan Vivekanandan Poongavanam, Ulrich Strych, Amadeo B. Biter, C. Patrick McAtee, Ebe A. Ewere, Christopher A. Seid |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
protein characterization Protein domain Pharmaceutical Science medicine.disease_cause Severe Acute Respiratory Syndrome Pichia Article Pichia pastoris law.invention 03 medical and health sciences Industrial Microbiology 0302 clinical medicine Protein Domains law protein purification Protein purification medicine Humans 030212 general & internal medicine Cloning Molecular Neutralizing antibody Coronavirus Vaccines Synthetic biology Antibody titer Viral Vaccines biology.organism_classification Virology hydrophobic interaction chromatography Recombinant Proteins 3. Good health circular dichroism Titer 030104 developmental biology Severe acute respiratory syndrome-related coronavirus 8. Economic growth Fermentation Spike Glycoprotein Coronavirus biology.protein Recombinant DNA |
| Zdroj: | Journal of Pharmaceutical Sciences Journal of pharmaceutical sciences |
| ISSN: | 0022-3549 |
| DOI: | 10.1016/j.xphs.2017.04.037 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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