Application of Second-Derivative Fluorescence Spectroscopy to Monitor Subtle Changes in a Monoclonal Antibody Structure

In this study, the tertiary structure of a monoclonal antibody was analyzed under thermal and chemical stresses using second‐derivative fluorescence spectroscopy. The effect of polyols, sucrose, and ethylene glycol on the tertiary structure of monoclonal antibody‐U (mAb‐U) (pH 7.0) was studied under thermal stress (25°C-75°C). The tertiary structure of mAb‐U was also analyzed upon chemical denaturation using urea (2.0-8.0 M). The second derivative of mAb‐U showed three bands corresponding to the three spectral classes of tryptophan, class I (330 nm), class II (340 nm), and class III (350 nm). Class II was higher in intensity in the presence of polyols compared with the solution without any polyol. Thermally denatured structure of mAb‐U in sucrose and ethylene glycol was distinctly different than that in buffer. Addition of urea resulted in a decrease in intensity of class I and II, and an increase in intensity of class III implying unfolding. This study showed that second‐derivative fluorescence spectroscopy is an effective tool to monitor subtle alterations in the tertiary structure of proteins. The unfolding of a protein is reflected as an increase in the intensity of the polar class III accompanied with a decrease in the intensity of class I.