Large-Scale Purification and Characterization of Recombinant Receptor-Binding Domain (RBD) of SARS-CoV-2 Spike Protein Expressed in Yeast

Abstract

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 <i>Pichia pastoris</i> 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 &gt;99% purity. The protein&rsquo;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 &times; 10^&minus;8 to 6.67 &times; 10^&minus;8, demonstrated a high affinity for the ACE2 receptor, revealing this protein as a promising candidate to prevent the entry of COVID-19 virus.