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 β-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.

Transforming growth factor alpha (TGFa) is an important epidermal growth factor receptor (EGFR) ligand. Over-expression of both molecules in epithelial tumors has been correlated with poor prognosis and disease progression. Due to the importance of TGFa in tumorigenesis, this molecule has great potential for cancer immunotherapy. We previously designed a TGFa-based vaccine consisting of a fusion protein between human TGFa (hTGFa) and P64k protein from Neisseria meningitidis expressed in Escherichia coli. However, this protein was obtained highly aggregated, which hampered its introduction into clinical use. In this study, we demonstrate that this aggregation state is not a consequence of IMAC purification, but is formed after bacterial disruption. To obtain this protein as a monomer, we designed a procedure that included an unfolding/refolding step at the end of purification. We verified that hTGFa in the refolded fusion protein (hTGFa-P64k-r) is immunogenic in mice. The latter was capable of inducing a humoral immune response against hTGFa identical to that generated with the aggregated fusion protein, demonstrating that the aggregation level has no influence on hTGFa immunogenicity. We also showed that TGFa-directed antibodies induced apoptosis in A431 cells. The present results also validated the potential use of this vaccine in cancer patients with tumors overexpressing TGFa. Drug Dev Res 69 : 481-494, 2008.