Conformational characterization of the mammalian-expressed SARS-CoV-2 recombinant receptor binding domain, a COVID-19 vaccine.

The COVID-19 pandemic has caused a large number of diseases worldwide. There are few vaccines to constrain this disease and the value of them is high. In this sense, the antigens of the vaccine platform Soberana, the receptor binding domain from SARS-CoV-2 Spike protein, both the monomeric (mRBD) and dimeric (dRBD) forms, have been developed. This study encompassed several analyses by different techniques like circular dichroism (CD), fluorescence spectroscopy (FS) and Gel Filtration- High Performance Liquid ChLC of mRBD and dRBD. Monomer and dimer exhibited similar far-UV CD spectral characteristics with 54% of ß-sheet content. Similar conformational features according to near-UV CD and FS studies were observed in both RBD. Stress stability studies by far-UV CD, FS, biological activity and GF-HPLC at 37 °C showed that mRBD is very stable. On the other hand, dRBD fluorescent emission showed a shift towards higher wavelengths as the incubation time increases, suggesting exposition of tryptophan residues, unlike what happens with mRBD. Biological activity outcome confirms these results. GF-HPLC profiles showed that in mRBD, the product of molecular stress are dimers and does not increase over time. However, dRBD showed dimer fragmentation as the main degradation species. This study reveals the usefulness of CD techniques for the analysis of degradation of RBD molecules as well as showed the difference in stability of both RBD molecules. Besides, our work provides useful insights into the production of a key protein used in diagnosis and therapeutics to fight COVID-19 pandemia.

Conformational characterization of the mammalian-expressed SARS-CoV-2 recombinant receptor binding domain, a COVID-19 vaccine

The COVID-19 pandemic has caused a large number of diseases worldwide. There are few vaccines to constrain this disease and the value of them is high. In this sense, the antigens of the vaccine platform Soberana, the receptor binding domain from SARS-CoV-2 Spike protein, both the monomeric (mRBD) and dimeric (dRBD) forms, have been developed. This study encompassed several analyses by different techniques like circular dichroism (CD), fluorescence spectroscopy (FS) and Gel Filtration- High Performance Liquid ChLC of mRBD and dRBD. Monomer and dimer exhibited similar far-UV CD spectral characteristics with 54% of ß-sheet content. Similar conformational features according to near-UV CD and FS studies were observed in both RBD. Stress stability studies by far-UV CD, FS, biological activity and GF-HPLC at 37 °C showed that mRBD is very stable. On the other hand, dRBD fluorescent emission showed a shift towards higher wavelengths as the incubation time increases, suggesting exposition of tryptophan residues, unlike what happens with mRBD. Biological activity outcome confirms these results. GF-HPLC profiles showed that in mRBD, the product of molecular stress are dimers and does not increase over time. However, dRBD showed dimer fragmentation as the main degradation species. This study reveals the usefulness of CD techniques for the analysis of degradation of RBD molecules as well as showed the difference in stability of both RBD molecules. Besides, our work provides useful insights into the production of a key protein used in diagnosis and therapeutics to fight COVID-19 pandemia.

Conformational characterization of a novel anti-HER2 candidate antibody.

Regulatory agencies establish that a broad physicochemical and biological characterization is necessary for the evaluation of comparability between a biosimilar candidate product and a reference commercial drug. Between them, conformational characterization of proteins is of vital importance to determine its folding and biological functions. In this work, the conformational features of a novel monoclonal antibody (called 5G4) were evaluated by means of circular dichroism spectroscopy and fluorescence. Secondary structure and thermal stability of mAbs were determined by circular dichroism in the far ultraviolet, while three-dimensional folding of proteins was analyzed by both circular dichroism in the near ultraviolet and intrinsic tryptophan fluorescence. In all experiments, Herceptin (Roche) was used as control. Both antibodies showed a composition of secondary structure predominantly of ß-sheets (55-56%) and thermal stability of ~ 75°C, suggesting structural similarity. The three-dimensional folding of proteins was also similar due to the absorption spectra of the aromatic residues and the emission wavelength maxima by fluorescence were comparable. The values of the fluorescence attenuation constant (Stern-Volmer constant) for increasing concentrations of acrylamide were also similar, suggesting a degree of exposure of tryptophan residues similar, although it was slightly decreased for Herceptin. Our data permit to consider that 5G4 monoclonal antibody showed similar conformational characteristics when compared with Herceptin.