Nagar Gaurav, Jain Siddharth, Rajurkar Meghraj, Lothe Rakesh, Rao Harish, Majumdar Sourav, Gautam Manish, Rodriguez-Aponte Sergio A, Crowell Laura E, Love J Christopher, Dandekar Prajakta, Puranik Amita, Gairola Sunil, Shaligram Umesh, Jain Ratnesh
Serum Institute of India Pvt. Ltd., Hadapsar, Pune 411028, India.
Department of Biological Engineering, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Vaccines (Basel). 2023 Oct 16;11(10):1602. doi: 10.3390/vaccines11101602.
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 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 to 6.67 × 10, demonstrated a high affinity for the ACE2 receptor, revealing this protein as a promising candidate to prevent the entry of COVID-19 virus.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）刺突蛋白是众多用于2019冠状病毒病（COVID-19）的基于蛋白质的疫苗的重要组成部分。该刺突蛋白的受体结合结构域是一种很有前景的抗原，易于在微生物宿主中表达，且能以相对较低的生产成本进行扩大生产。本研究从商业化角度描述了目前正在进行临床试验的SARS-CoV-2蛋白受体结合结构域（RBD）的生产、纯化和特性分析。该蛋白在一个1200升容量的大型生物反应器中表达。通过混合模式色谱随后进行疏水相互作用色谱来进行蛋白捕获和纯化。这个两步纯化过程产生的RBD总体生产率约为21毫克/升，纯度大于99%。还分别使用基于液相色谱-质谱联用（LCMS）的肽图谱分析、圆二色光谱和荧光光谱对该蛋白的一级、二级和三级结构进行了验证。对该糖蛋白的糖基化、分子量、纯度、二硫键等质量属性进行了进一步表征。通过结构分析，证实该产品在大规模生产过程中不同批次间保持了一致的质量。还使用人血管紧张素转换酶2受体评估了刺突蛋白RBD的结合能力。KD值的低结合常数范围在3.63×10至6.67×10之间，表明对ACE2受体具有高亲和力，揭示该蛋白是预防COVID-19病毒进入的一个有前景的候选物。
