| Autor: |
Nagar G; Serum Institute of India Pvt. Ltd., Hadapsar, Pune 411028, India., Jain S; Serum Institute of India Pvt. Ltd., Hadapsar, Pune 411028, India., Rajurkar M; Serum Institute of India Pvt. Ltd., Hadapsar, Pune 411028, India., Lothe R; Serum Institute of India Pvt. Ltd., Hadapsar, Pune 411028, India., Rao H; Serum Institute of India Pvt. Ltd., Hadapsar, Pune 411028, India., Majumdar S; Serum Institute of India Pvt. Ltd., Hadapsar, Pune 411028, India., Gautam M; Serum Institute of India Pvt. Ltd., Hadapsar, Pune 411028, India., Rodriguez-Aponte SA; Department of Biological Engineering, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA., Crowell LE; Department of Chemical Engineering, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA., Love JC; Department of Chemical Engineering, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA., Dandekar P; Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, Matunga, Mumbai 400019, India., Puranik A; Department of Biological Sciences and Biotechnology, Institute of Chemical Technology, Matunga, Mumbai 400019, India., Gairola S; Serum Institute of India Pvt. Ltd., Hadapsar, Pune 411028, India., Shaligram U; Serum Institute of India Pvt. Ltd., Hadapsar, Pune 411028, India., Jain R; Department of Biological Sciences and Biotechnology, Institute of Chemical Technology, Matunga, Mumbai 400019, India. |
| Abstrakt: |
SARS-CoV-2 spike protein is an essential component of numerous protein-based vaccines for COVID-19. The receptor-binding domain of this spike protein is a promising antigen with ease of expression in microbial hosts and scalability at comparatively low production costs. This study describes the production, purification, and characterization of RBD of SARS-CoV-2 protein, which is currently in clinical trials, from a commercialization perspective. The protein was expressed in Pichia pastoris in a large-scale bioreactor of 1200 L capacity. Protein capture and purification are conducted through mixed-mode chromatography followed by hydrophobic interaction chromatography. This two-step purification process produced RBD with an overall productivity of ~21 mg/L at >99% purity. The protein's primary, secondary, and tertiary structures were also verified using LCMS-based peptide mapping, circular dichroism, and fluorescence spectroscopy, respectively. The glycoprotein was further characterized for quality attributes such as glycosylation, molecular weight, purity, di-sulfide bonding, etc. Through structural analysis, it was confirmed that the product maintained a consistent quality across different batches during the large-scale production process. The binding capacity of RBD of spike protein was also assessed using human angiotensin-converting enzyme 2 receptor. A low binding constant range of KD values, ranging between 3.63 × 10 -8 to 6.67 × 10 -8 , demonstrated a high affinity for the ACE2 receptor, revealing this protein as a promising candidate to prevent the entry of COVID-19 virus. |
