SARS-CoV-2 antigens: Impact of bioprocess

Serological assays are valuable tools to study the spread of SARS-CoV-2 and, more importantly, to identify people who were already infected and potentially immune to reinfection with the virus. The SARS-CoV-2 Spike protein and its receptor binding domain (RBD) are the antigens with the greatest potential for developing SARS-CoV-2 serological assays.

Furthermore, structural studies of these antigens are key to understanding the molecular basis of Spike's interaction with the angiotensin-converting enzyme 2 receptor, which will hopefully enable the development of treatments for COVID-19. Therefore, it is urgent that significant amounts of this protein be made available in the highest quality. In this study, we produced Spike and RBD in two human-derived cell hosts: HEK293-E6 and Expi293F™.

We evaluated the impact of different and scalable bioprocessing approaches on Spike and RBD production yields and, more importantly, on the quality attributes of these antigens. Using negative and positive sera collected from human donors, we show excellent performance of the produced antigens, evaluated in serological enzyme-linked immunosorbent assays (ELISA), as indicated by the high specificity and sensitivity of the test. We show robust Spike productions with final yields of approx.

2 mg/L of culture that were maintained regardless of production scale or cell culture strategy. To our knowledge, the final yield of 90  mg/L of culture obtained for RBD production was the highest reported to date. An in-depth characterization of the SARS-CoV-2 Spike and RBD proteins was performed, namely the oligomeric state of the antigen, glycosylation profiles, and thermal stability during storage.

Correlation of these quality attributes with ELISA performance shows equivalent reactivity to SARS-CoV-2 positive serum, for all Spike and RBD produced, and for all storage conditions tested. Overall, we provide simple protocols to produce high-quality SARS-CoV-2 Spike and RBD antigens, which can be easily adapted to academic and industrial settings; and integrating, for the first time, bioprocess impact studies with in-depth characterization of these proteins, correlating antigen glycosylation and biophysical attributes with the performance of COVID-19 serological tests.