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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.

The expression and production of pharmaceutical, industrial and veterinary proteins in plants is an attractive approach. These expression hosts bear an enormous production potential in terms of volume, easy processing of the starting material, and safety of the final product due to the lack of pathogens able to infect animal and human cells. Antibodies are among the most frequently proteins expressed in plants, subsequently called plantibodies. However, plantibodies are differently glycosylated in plant cells, with oligosaccharide residues being added which may them immunogenic in the final organism. For that reason, several strategies have been developed to genetically modify host plants to mimic the N-glycosylation patterns typical in animal cells. This work was aimed at developing a strategy to obtain a aglycosylated plantibody version of nimotuzumab, the first antibody registered as a product in Cuba for cancer immunotherapy. The strategy comprised the genetic modification of the heavy chain glycosylation site of nimotuzumab, and its expression as an aglycosylated protein in tobacco leaves, by means of developing a transient expression system using Agrobacterium infiltration into tobacco leaves for the initial characterization of the plantibody. It was demonstrated that transgenic plants were capable of producing a plant-derived nimotuzumab antibody which retained the antitumor activity in vitro and in vivo, compared to its glycosylated counterpart produced in mammalian cells. This work demonstrates the potential of transgenic plants to produce aglycosylated therapeutic antibodies for cancer treatment, and won the National Award of the Academy of Sciences of Cuba in 2012.

The Epidermal Growth Factor Receptor (EGFR) plays an essential role in regulating neoplastic processes. EGFR over-expression in many human epithelial tumors has been correlated with disease progression and bad prognosis. Passive EGFR-directed immunotherapy has been introduced in medical oncology practice, but no active specific approaches have been used. We designed a vaccine candidate based on the extracellular domain (ECD) of human EGFR (HER1), and the homologous vaccine for mice based on murine EGFR. This vaccine candidate uses the Very Small-Sized Proteoliposomes from Neisseria meningitidis (VSSP) and Montanide ISA 51-VG as adjuvants. The autologous vaccine circumvents the tolerance to self EGFR by inducing a specific immune response with an anti-metastatic effect on EGFR+ tumors. The vaccine candidate based on HER1-ECD induced anti-EGFR polyclonal antibodies (PAb) that bind EGFR+ human tumor cell lines from different tissues. These anti-EGFR PAb abrogate ligand-dependent EGFR phosphorylation, provoking the inhibition of tumor cell growth and apoptosis. Preclinical results further encouraged the evaluation of the HER1 vaccine candidate in phase I clinical trials

The Epidermal Growth Factor Receptor (EGFR) plays an essential role in regulating neoplastic processes. EGFR over-expression in many human epithelial tumors has been correlated with disease progression and bad prognosis. Passive EGFR-directed immunotherapy has been introduced in medical oncology practice, but no active specific approaches have been used. We designed a vaccine candidate based on the extracellular domain (ECD) of human EGFR (HER1), and the homologous vaccine for mice based on murine EGFR. This vaccine candidate uses the Very Small-Sized Proteoliposomes from Neisseria meningitidis (VSSP) and Montanide ISA 51-VG as adjuvants. The autologous vaccine circumvents the tolerance to self EGFR by inducing a specific immune response with an anti-metastatic effect on EGFR+ tumors. The vaccine candidate based on HER1-ECD induced anti-EGFR polyclonal antibodies (PAb) that bind EGFR+ human tumor cell lines from different tissues. These anti-EGFR PAb abrogate ligand-dependent EGFR phosphorylation, provoking the inhibition of tumor cell growth and apoptosis. Preclinical results further encouraged the evaluation of the HER1 vaccine candidate in phase I clinical trials