Large-Scale Purification and Characterization of Recombinant Receptor-Binding Domain (RBD) of SARS-CoV-2 Spike Protein Expressed in Yeast
Name
vaccines-11-01602.pdf
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5.75 MB
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Checksum (MD5)
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Author(s)
Nagar, Gaurav
Jain, Siddharth
Rajurkar, Meghraj
Lothe, Rakesh
Rao, Harish
Majumdar, Sourav
Gautam, Manish
Rodriguez-Aponte, Sergio A.
Crowell, Laura E.
Love, J. Christopher
Date Issued
October 16, 2023
Publisher
Multidisciplinary Digital Publishing Institute
Citation
Vaccines 11 (10): 1602 (2023)
Version
Final published version
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 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.
MIT Department
Massachusetts Institute of Technology. Department of Biological Engineering
Koch Institute for Integrative Cancer Research at MIT
Massachusetts Institute of Technology. Department of Chemical Engineering
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DOI of Published Version
https://doi.org/10.3390/vaccines11101602
