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Development of a scalable single process for producing SARS-CoV-2 RBD monomer and dimer vaccine antigens. 2023
Tammy Boggiano-Ayo, and
Julio Palacios-Oliva, and
Sumlai Lozada-Chang, and
Ernesto Relova-Hernandez, and
Jose Gomez-Perez, and
Gonzalo Oliva, and
Lourdes Hernandez, and
Alexi Bueno-Soler, and
Daidee Montes de Oca, and
Osvaldo Mora, and
Roberto Machado-Santisteban, and
Dayana Perez-Martinez, and
Beatriz Perez-Masson, and
Yanelys Cabrera Infante, and
Lisandra Calzadilla-Rosado, and
Yaima Ramirez, and
Judey Aymed-Garcia, and
Ingrid Ruiz-Ramirez, and
Yamile Romero, and
Tania Gomez, and
Luis A Espinosa, and
Luis Javier Gonzalez, and
Annia Cabrales, and
Osmany Guirola, and
Kathya Rashida de la Luz, and
Franciscary Pi-Estopiñan, and
Belinda Sanchez-Ramirez, and
Dagmar Garcia-Rivera, and
Yuri Valdes-Balbin, and
Gertrudis Rojas, and
Kalet Leon-Monzon, and
Eduardo Ojito-Magaz, and
Eugenio Hardy
Process Development Direction, Center of Molecular Immunology, Havana, Cuba.
We have developed a single process for producing two key COVID-19 vaccine antigens: SARS-CoV-2 receptor binding domain (RBD) monomer and dimer. These antigens are featured in various COVID-19 vaccine formats, including SOBERANA 01 and the licensed SOBERANA 02, and SOBERANA Plus. Our approach involves expressing RBD (319-541)-His6 in Chinese hamster ovary (CHO)-K1 cells, generating and characterizing oligoclones, and selecting the best RBD-producing clones. Critical parameters such as copper supplementation in the culture medium and cell viability influenced the yield of RBD dimer. The purification of RBD involved standard immobilized metal ion affinity chromatography (IMAC), ion exchange chromatography, and size exclusion chromatography. Our findings suggest that copper can improve IMAC performance. Efficient RBD production was achieved using small-scale bioreactor cell culture (2 L). The two RBD forms - monomeric and dimeric RBD - were also produced on a large scale (500 L). This study represents the first large-scale application of perfusion culture for the production of RBD antigens. We conducted a thorough analysis of the purified RBD antigens, which encompassed primary structure, protein integrity, N-glycosylation, size, purity, secondary and tertiary structures, isoform composition, hydrophobicity, and long-term stability. Additionally, we investigated RBD-ACE2 interactions, in vitro ACE2 recognition of RBD, and the immunogenicity of RBD antigens in mice. We have determined that both the monomeric and dimeric RBD antigens possess the necessary quality attributes for vaccine production. By enabling the customizable production of both RBD forms, this unified manufacturing process provides the required flexibility to adapt rapidly to the ever-changing demands of emerging SARS-CoV-2 variants and different COVID-19 vaccine platforms.
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