Development of a scalable single process for producing SARS-CoV-2 RBD monomer and dimer vaccine antigens

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 involve...

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Main Authors: Tammy Boggiano-Ayo, Julio Palacios-Oliva, Sumlai Lozada-Chang, Ernesto Relova-Hernandez, Jose Gomez-Perez, Gonzalo Oliva, Lourdes Hernandez, Alexi Bueno-Soler, Daidee Montes de Oca, Osvaldo Mora, Roberto Machado-Santisteban, Dayana Perez-Martinez, Beatriz Perez-Masson, Yanelys Cabrera Infante, Lisandra Calzadilla-Rosado, Yaima Ramirez, Judey Aymed-Garcia, Ingrid Ruiz-Ramirez, Yamile Romero, Tania Gomez, Luis A. Espinosa, Luis Javier Gonzalez, Annia Cabrales, Osmany Guirola, Kathya Rashida de la Luz, Franciscary Pi-Estopiñan, Belinda Sanchez-Ramirez, Dagmar Garcia-Rivera, Yuri Valdes-Balbin, Gertrudis Rojas, Kalet Leon-Monzon, Eduardo Ojito-Magaz, Eugenio Hardy
Format: Article
Language:English
Published: Frontiers Media S.A. 2023-11-01
Series:Frontiers in Bioengineering and Biotechnology
Subjects:
Online Access:https://www.frontiersin.org/articles/10.3389/fbioe.2023.1287551/full
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author Tammy Boggiano-Ayo
Julio Palacios-Oliva
Sumlai Lozada-Chang
Ernesto Relova-Hernandez
Jose Gomez-Perez
Gonzalo Oliva
Lourdes Hernandez
Alexi Bueno-Soler
Daidee Montes de Oca
Osvaldo Mora
Roberto Machado-Santisteban
Dayana Perez-Martinez
Beatriz Perez-Masson
Yanelys Cabrera Infante
Lisandra Calzadilla-Rosado
Yaima Ramirez
Judey Aymed-Garcia
Ingrid Ruiz-Ramirez
Yamile Romero
Tania Gomez
Luis A. Espinosa
Luis Javier Gonzalez
Annia Cabrales
Osmany Guirola
Kathya Rashida de la Luz
Franciscary Pi-Estopiñan
Belinda Sanchez-Ramirez
Dagmar Garcia-Rivera
Yuri Valdes-Balbin
Gertrudis Rojas
Kalet Leon-Monzon
Eduardo Ojito-Magaz
Eugenio Hardy
author_facet Tammy Boggiano-Ayo
Julio Palacios-Oliva
Sumlai Lozada-Chang
Ernesto Relova-Hernandez
Jose Gomez-Perez
Gonzalo Oliva
Lourdes Hernandez
Alexi Bueno-Soler
Daidee Montes de Oca
Osvaldo Mora
Roberto Machado-Santisteban
Dayana Perez-Martinez
Beatriz Perez-Masson
Yanelys Cabrera Infante
Lisandra Calzadilla-Rosado
Yaima Ramirez
Judey Aymed-Garcia
Ingrid Ruiz-Ramirez
Yamile Romero
Tania Gomez
Luis A. Espinosa
Luis Javier Gonzalez
Annia Cabrales
Osmany Guirola
Kathya Rashida de la Luz
Franciscary Pi-Estopiñan
Belinda Sanchez-Ramirez
Dagmar Garcia-Rivera
Yuri Valdes-Balbin
Gertrudis Rojas
Kalet Leon-Monzon
Eduardo Ojito-Magaz
Eugenio Hardy
author_sort Tammy Boggiano-Ayo
collection DOAJ
description 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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spelling doaj.art-e0706f0426fc462d821685ef6ce26f542023-11-18T10:20:33ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852023-11-011110.3389/fbioe.2023.12875511287551Development of a scalable single process for producing SARS-CoV-2 RBD monomer and dimer vaccine antigensTammy Boggiano-Ayo0Julio Palacios-Oliva1Sumlai Lozada-Chang2Ernesto Relova-Hernandez3Jose Gomez-Perez4Gonzalo Oliva5Lourdes Hernandez6Alexi Bueno-Soler7Daidee Montes de Oca8Osvaldo Mora9Roberto Machado-Santisteban10Dayana Perez-Martinez11Beatriz Perez-Masson12Yanelys Cabrera Infante13Lisandra Calzadilla-Rosado14Yaima Ramirez15Judey Aymed-Garcia16Ingrid Ruiz-Ramirez17Yamile Romero18Tania Gomez19Luis A. Espinosa20Luis Javier Gonzalez21Annia Cabrales22Osmany Guirola23Kathya Rashida de la Luz24Franciscary Pi-Estopiñan25Belinda Sanchez-Ramirez26Dagmar Garcia-Rivera27Yuri Valdes-Balbin28Gertrudis Rojas29Kalet Leon-Monzon30Eduardo Ojito-Magaz31Eugenio Hardy32Process Development Direction, Center of Molecular Immunology, Havana, CubaProcess Development Direction, Center of Molecular Immunology, Havana, CubaProcess Development Direction, Center of Molecular Immunology, Havana, CubaImmunology and Immunobiology Direction, Center of Molecular Immunology, Havana, CubaQuality Direction, Center of Molecular Immunology, Havana, CubaProcess Direction, Center of Molecular Immunology, Havana, CubaProcess Direction, Center of Molecular Immunology, Havana, CubaProcess Development Direction, Center of Molecular Immunology, Havana, CubaProcess Development Direction, Center of Molecular Immunology, Havana, CubaProcess Direction, Center of Molecular Immunology, Havana, CubaProcess Development Direction, Center of Molecular Immunology, Havana, CubaImmunology and Immunobiology Direction, Center of Molecular Immunology, Havana, CubaImmunology and Immunobiology Direction, Center of Molecular Immunology, Havana, CubaImmunology and Immunobiology Direction, Center of Molecular Immunology, Havana, CubaProcess Development Direction, Center of Molecular Immunology, Havana, CubaImmunology and Immunobiology Direction, Center of Molecular Immunology, Havana, CubaImmunology and Immunobiology Direction, Center of Molecular Immunology, Havana, CubaQuality Direction, Center of Molecular Immunology, Havana, CubaImmunology and Immunobiology Direction, Center of Molecular Immunology, Havana, CubaQuality Direction, Center of Molecular Immunology, Havana, CubaCenter for Genetic Engineering and Biotechnology, Playa, CubaCenter for Genetic Engineering and Biotechnology, Playa, CubaCenter for Genetic Engineering and Biotechnology, Playa, CubaCenter for Genetic Engineering and Biotechnology, Playa, CubaProcess Development Direction, Center of Molecular Immunology, Havana, CubaImmunology and Immunobiology Direction, Center of Molecular Immunology, Havana, CubaImmunology and Immunobiology Direction, Center of Molecular Immunology, Havana, CubaFinlay Vaccine Institute, Havana, CubaFinlay Vaccine Institute, Havana, CubaImmunology and Immunobiology Direction, Center of Molecular Immunology, Havana, CubaImmunology and Immunobiology Direction, Center of Molecular Immunology, Havana, CubaProcess Development Direction, Center of Molecular Immunology, Havana, CubaProcess Development Direction, Center of Molecular Immunology, Havana, CubaWe 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.https://www.frontiersin.org/articles/10.3389/fbioe.2023.1287551/fullSARS-CoV-2RBDCHO cellsperfusion cultureCOVID-19vaccine
spellingShingle Tammy Boggiano-Ayo
Julio Palacios-Oliva
Sumlai Lozada-Chang
Ernesto Relova-Hernandez
Jose Gomez-Perez
Gonzalo Oliva
Lourdes Hernandez
Alexi Bueno-Soler
Daidee Montes de Oca
Osvaldo Mora
Roberto Machado-Santisteban
Dayana Perez-Martinez
Beatriz Perez-Masson
Yanelys Cabrera Infante
Lisandra Calzadilla-Rosado
Yaima Ramirez
Judey Aymed-Garcia
Ingrid Ruiz-Ramirez
Yamile Romero
Tania Gomez
Luis A. Espinosa
Luis Javier Gonzalez
Annia Cabrales
Osmany Guirola
Kathya Rashida de la Luz
Franciscary Pi-Estopiñan
Belinda Sanchez-Ramirez
Dagmar Garcia-Rivera
Yuri Valdes-Balbin
Gertrudis Rojas
Kalet Leon-Monzon
Eduardo Ojito-Magaz
Eugenio Hardy
Development of a scalable single process for producing SARS-CoV-2 RBD monomer and dimer vaccine antigens
Frontiers in Bioengineering and Biotechnology
SARS-CoV-2
RBD
CHO cells
perfusion culture
COVID-19
vaccine
title Development of a scalable single process for producing SARS-CoV-2 RBD monomer and dimer vaccine antigens
title_full Development of a scalable single process for producing SARS-CoV-2 RBD monomer and dimer vaccine antigens
title_fullStr Development of a scalable single process for producing SARS-CoV-2 RBD monomer and dimer vaccine antigens
title_full_unstemmed Development of a scalable single process for producing SARS-CoV-2 RBD monomer and dimer vaccine antigens
title_short Development of a scalable single process for producing SARS-CoV-2 RBD monomer and dimer vaccine antigens
title_sort development of a scalable single process for producing sars cov 2 rbd monomer and dimer vaccine antigens
topic SARS-CoV-2
RBD
CHO cells
perfusion culture
COVID-19
vaccine
url https://www.frontiersin.org/articles/10.3389/fbioe.2023.1287551/full
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