Production of high-quality SARS-CoV-2 antigens: Impact of bioprocess and storage on glycosylation, biophysical attributes, and ELISA serologic tests performance

Rute Castro, Lígia S. Nobre, Rute P. Eleutério, Mónica Thomaz, António Pires, Sandra M. Monteiro, Sónia Mendes, Ricardo A. Gomes, João J. Clemente, Marcos F.Q. Sousa, Filipe Pinto, Ana C. Silva, Micael C. Freitas, Ana R. Lemos, Onome Akpogheneta, Lindsay Kosack, Marie Louise Bergman, Nadia Duarte, Paula Matoso, Júlia CostaTiago M. Bandeiras, Patricia Gomes-Alves, Carlos P. Gonçalves, Jocelyne Demengeot, Paula M. Alves

Research output: Contribution to journalArticlepeer-review

24 Citations (Scopus)


Serological assays are valuable tools to study SARS-CoV-2 spread and, importantly, to identify individuals that were already infected and would be potentially immune to a virus reinfection. SARS-CoV-2 Spike protein and its receptor binding domain (RBD) are the antigens with higher potential to develop SARS-CoV-2 serological assays. Moreover, structural studies of these antigens are key to understand the molecular basis for Spike interaction with angiotensin converting enzyme 2 receptor, hopefully enabling the development of COVID-19 therapeutics. Thus, it is urgent that significant amounts of this protein became available at 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 these antigens' quality attributes. Using negative and positive sera collected from human donors, we show an excellent performance of the produced antigens, assessed in serologic enzyme-linked immunosorbent assay (ELISA) tests, as denoted 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 independently of the production scale or cell culture strategy. To the best of 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 SARS-CoV-2 Spike and RBD proteins was performed, namely the antigen's oligomeric state, glycosylation profiles, and thermal stability during storage. The correlation of these quality attributes with ELISA performance show equivalent reactivity to SARS-CoV-2 positive serum, for all Spike and RBD produced, and for all storage conditions tested. Overall, we provide straightforward protocols to produce high-quality SARS-CoV-2 Spike and RBD antigens, that can be easily adapted to both academic and industrial settings; and integrate, for the first time, studies on the impact of bioprocess with an in-depth characterization of these proteins, correlating antigen's glycosylation and biophysical attributes to performance of COVID-19 serologic tests.

Original languageEnglish
Pages (from-to)2202-2219
Number of pages18
JournalBiotechnology and Bioengineering
Issue number6
Publication statusPublished - Jun 2021


  • bioprocess
  • COVID-19
  • glycosylation
  • production and purification
  • RBD
  • SARS-CoV-2
  • serologic assay
  • Spike
  • thermal stability during storage


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