ABSTRACT
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.
ABSTRACT
BACKGROUND: SOBERANA 02 is a COVID-19 vaccine based on SARS-CoV-2 recombinant RBD conjugated to tetanus toxoid (TT). SOBERANA Plus antigen is dimeric-RBD. Here we report safety and immunogenicity from phase I and IIa clinical trials using two-doses of SOBERANA 02 and three-doses (homologous) or heterologous (with SOBERANA Plus) protocols. METHOD: We performed an open-label, sequential and adaptive phase I to evaluate safety and explore the immunogenicity of SOBERANA 02 in two formulations (15 or 25 µg RBD-conjugated to 20 µg of TT) in 40 subjects, 19-59-years-old. Phase IIa was open-label including 100 volunteers 19-80-years, receiving two doses of SOBERANA 02-25 µg. In both trials, half of volunteers were selected to receive a third dose of the corresponding SOBERANA 02 and half received a heterologous dose of SOBERANA Plus. Primary outcome was safety. The secondary outcome was immunogenicity evaluated by anti-RBD IgG ELISA, molecular neutralization of RBD:hACE2 interaction, live-virus-neutralization and specific T-cells response. RESULTS: The most frequent adverse event (AE) was local pain, other AEs had frequencies ≤ 5%. No serious related-AEs were reported. Phase IIa confirmed the safety in 60 to 80-years-old subjects. In phase-I SOBERANA 02-25 µg elicited higher immune response than SOBERANA 02-15 µg and progressed to phase IIa. Phase IIa results confirmed the immunogenicity of SOBERANA 02-25 µg even in 60-80-years. Two doses of SOBERANA02-25 µg elicited an immune response similar to that of the Cuban Convalescent Serum Panel and it was higher after the homologous and heterologous third doses. The heterologous scheme showed a higher immunological response. Anti-RBD IgG neutralized the delta variant in molecular assay, with a 2.5-fold reduction compared to D614G neutralization. CONCLUSIONS: SOBERANA 02 was safe and immunogenic in persons aged 19-80 years, eliciting neutralizing antibodies and specific T-cell response. Highest immune responses were obtained in the heterologous three doses protocol. TRIAL REGISTRY: https://rpcec.sld.cu/trials/RPCEC00000340, https://rpcec.sld.cu/trials/RPCEC00000347.
Subject(s)
COVID-19 Vaccines , COVID-19 , Adult , Aged , Aged, 80 and over , Antibodies, Neutralizing , Antibodies, Viral , COVID-19/prevention & control , COVID-19/therapy , COVID-19 Vaccines/adverse effects , Humans , Immunization, Passive , Immunogenicity, Vaccine , Immunoglobulin G , Middle Aged , SARS-CoV-2 , Young Adult , COVID-19 SerotherapyABSTRACT
En este trabajo se presenta la aplicación del Análisis de Componentes Principales, mediante el programa THE UNSCRAMBLER versión 8.0, a los datos registrados en un período de 2 años en la etapa de purificación de una planta de producción de Eritropoyetina Humana Recombinante que está basada en varios pasos cromatográficos, de forma similar a los procesos de purificación de proteínas recombinantes que se utilizan como vacunas preventivas o terapéuticas. Se logró reducir dimensionalidad al obtenerse dos componentes principales que explican el 81 por ciento de la varianza de 18 variables originales relacionadas con cuatro pasos cromatográficos. Como resultado se llegó a definir cuáles son las variables que mayor aporte tienen a la variabilidad del proceso en la etapa de purificación, permitiendo extraer información útil para lograr un mayor entendimiento del proceso y enriquecer las estrategias de control en la planta. Dichos resultados corroboraron experiencias prácticas de especialistas de la planta y permitieron dar recomendaciones a considerar en el plan de verificación continuada del proceso como proponer cinco variables como controles de proceso y tener en cuenta que el rendimiento del segundo paso cromatográfico es el más influyente de los rendimientos considerados en la variabilidad(AU)
This paper presents the application of the Principal Component Analysis, using the program THE UNSCRAMBLER version 8.0, to the data recorded during two years in the purification stage of a Recombinant Human Erythropoietin plant that is based on several chromatographic steps, similar to the purification process of recombinant proteins that are used as preventive or therapeutic vaccines. Dimensionality was reduced by obtaining two main components that explain 81 percent of the variance of 18 original variables related to four chromatographic steps. As a result, it was possible to define which variables have the greatest contribution to the variability of the process in the purification stage, allowing to extract useful information to achieve a greater understanding of the process and enrich the control strategies in the plant. These results corroborated practical experiences of plant specialists and allowed for recommendations to be considered in the continuous verification plan of the process, such as proposing three variables as process controls and taking into account that the performance of the second step is the most influential of the performances considered in the variability(AU)
Subject(s)
Humans , Biological Products/therapeutic use , Chromatography/methods , Principal Component Analysis/methods , Reference Drugs , BiopharmaceuticsABSTRACT
Introducción: La evaluación de los factores claves de éxito de la innovación en la industria biotecnológica aplicada a la salud constituye un problema de investigación. Objetivo: Evaluar la innovación en un centro cubano de la biotecnología aplicada a la salud. Métodos: Se aplicó en dos momentos la encuesta de innovación en el Centro de Inmunología Molecular. Los resultados se procesaron con el diagrama de caja, la estadística básica y el análisis de la correlación. Se analizaron artículos científicos que muestran los resultados de la innovación en el centro. Resultados: La encuesta se aplicó en el 2015 y 2018 a 33 miembros del consejo de dirección y a especialistas, identificándose como estrategias genéricas las de innovación, calidad y liderazgo y como objetivos de innovación los de calidad y el mercado. Las fuentes de innovación más importantes se refieren a las actividades de investigación y desarrollo, la producción, la alta dirección y los centros de investigaciones. Los factores que limitan están relacionados con el período largo de rentabilidad de la innovación, el financiamiento y la adquisición de los insumos. Conclusiones: La evaluación de la innovación en el centro de biotecnología corroboró la significación que tiene para la empresa la estrategia de innovación como factor clave, que lo ubica en una posición de excelencia en la industria biofarmacéutica cubana e internacional(AU)
Introduction: The assessment of the key success factors of innovation in the biotechnology industry applied to health constitutes a research problem. Objective: To assess innovation process in a Cuban center of biotechnology applied to health. Methods: It was applied in two moments the survey of innovation in the Molecular Immunology Center. The results were processed by the box plot, basic statistics and analysis of the correlation. Scientific articles that show the results of innovation in the Center were analyzed. Results: The survey was applied in 2015 and 2018 to 33 members of the board of directors and to specialists, and being identified innovation, quality and leadership as generic strategies, and quality and the market as objectives of innovation. The most important sources of innovation referred to the research and development activities, production, top management and research centers. The limiting factors are related to the long term return of innovation, financing, and the acquisition of supplies. Conclusions: The assessment of innovation in the Biotechnology Center corroborated the significance of the innovation strategy for the company as a key factor, which places it in a position of excellence in the Cuban and international biopharmaceutical industry(AU)
Subject(s)
Biopharmaceutics , Biotechnology , InventionsABSTRACT
RESUMEN Introducción: La evaluación de los factores claves de éxito de la innovación en la industria biotecnológica aplicada a la salud constituye un problema de investigación. Objetivo: Evaluar la innovación en un centro cubano de la biotecnología aplicada a la salud. Métodos: Se aplicó en dos momentos la encuesta de innovación en el Centro de Inmunología Molecular. Los resultados se procesaron con el diagrama de caja, la estadística básica y el análisis de la correlación. Se analizaron artículos científicos que muestran los resultados de la innovación en el centro. Resultados: La encuesta se aplicó en el 2015 y 2018 a 33 miembros del consejo de dirección y a especialistas, identificándose como estrategias genéricas las de innovación, calidad y liderazgo y como objetivos de innovación los de calidad y el mercado. Las fuentes de innovación más importantes se refieren a las actividades de investigación y desarrollo, la producción, la alta dirección y los centros de investigaciones. Los factores que limitan están relacionados con el período largo de rentabilidad de la innovación, el financiamiento y la adquisición de los insumos. Conclusiones: La evaluación de la innovación en el centro de biotecnología corroboró la significación que tiene para la empresa la estrategia de innovación como factor clave, que lo ubica en una posición de excelencia en la industria biofarmacéutica cubana e internacional.
ABSTRACT Introduction: The assessment of the key success factors of innovation in the biotechnology industry applied to health constitutes a research problem. Objective: To assess innovation process in a Cuban center of biotechnology applied to health. Methods: It was applied in two moments the survey of innovation in the Molecular Immunology Center. The results were processed by the box plot, basic statistics and analysis of the correlation. Scientific articles that show the results of innovation in the Center were analyzed. Results: The survey was applied in 2015 and 2018 to 33 members of the board of directors and to specialists, and being identified innovation, quality and leadership as generic strategies, and quality and the market as objectives of innovation. The most important sources of innovation referred to the research and development activities, production, top management and research centers. The limiting factors are related to the long term return of innovation, financing, and the acquisition of supplies. Conclusions: The assessment of innovation in the Biotechnology Center corroborated the significance of the innovation strategy for the company as a key factor, which places it in a position of excellence in the Cuban and international biopharmaceutical industry.
