Conformational characterization of a novel anti-HER2 candidate antibody

Abstract

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.

Tools and methods for circular dichroism spectroscopy of proteins: a tutorial review

“…The pharmaceutical industry relies on CD to ensure consistency between batches of drugs both during development and production. Perez et al , 43 characterised a novel monoclonal antibody by near UV CD, far UV CD, and fluorescence spectroscopies. Secondary structure analyses using the DichroWeb 16 server, after minimizing the differences in protein concentration between batches by scaling, then enabled comparisons using DichroMatch.…”

Analytical Similarity Assessment of Biosimilars: Global Regulatory Landscape, Recent Studies and Major Advancements in Orthogonal Platforms

“…Near UV CD (250-300 nm) and fluorescence (FLR) spectroscopy have been traditionally the methods of choice for tertiary structure determination. In biosimilarity studies, these two techniques have been used either individually or together (Moro Pérez et al, 2019;Zhang et al, 2020). However, there have been limited studies involving the use of X-ray crystallography (XRC), a traditional technique for protein structural studies at the near-atomic level, as it works well only with proteins that can be crystallized, it requires expensive instrumentation, trained operators, and extensive time for analysis (Lerch et al, 2020).…”

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“…However, trastuzumab is produced using the Chinese Hamster Ovary (CHO) cells. Although similar conformational characteristics of 5G4 mAb and Herceptin ® were previously published [8], diff erences in the expression system could aff ect both the binding properties and biological activity of mAb 5G4, due to changes in the glycosylation pattern.…”

“…In this context, a 5G4 mAb was generated at the Center of Molecular Immunology (Havana, Cuba). Th e 5G4 mAb was generated by transferring a plasmid DNA encoding trastuzumab and produced by the non-secretory murine myeloma cells (NSO) expression system [8]. However, trastuzumab is produced using the Chinese Hamster Ovary (CHO) cells.…”