Biophysical study of bevacizumab structure and bioactivity under thermal and pH-stresses

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The evaluation of the structural stability and bioactivity of monoclonal antibodies (mAb) is a crucial step in the development of mAb therapeutic based products, since immunogenicity needs to be avoided. In the present work, a study was carried out to understand the changes on the structure and bioactivity of mAbs induced by different pH and temperature values. Structural changes of bevacizumab were monitored using fluorescence spectroscopy, circular dichroism (CD) and Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR). The secondary and tertiary structural content was monitored at six different pH values and at room temperature, upon heating up to 85 °C and upon cooling down to 20 °C. Furthermore, the temperature induced conformational changes were continuously monitored from 20 °C to 85 °C using fluorescence spectroscopy and circular dichroism, allowing to monitor the melting temperature of the protein at different pH values. The results showed that the thermal denaturation of bevacizumab was irreversible at all pH value. The conformational changes induced by pH were higher at extreme pH values (5, 9 and 10) than neutral pH. Thermal stability studies showed that pH 6 was the pH that confer bevacizumab the highest structural stability. These studies were confirmed by in vitro studies, where bevacizumab's bioactivity was measured by cell viability/proliferation at all pH values at room temperature, and it was found a higher bioactivity for pH 6. Biophysical and biological studies were correlated in order to understand the importance of the modifications in bevacizumab structural content on its bioactivity. However, a decrease in bevacizumab's bioactivity was observed for pH 8, 9 and 10. Overall, this work demonstrated the usefulness of the spectroscopy techniques for estimating the stability of therapeutic mAb during formulation development.

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