Parameter optimization for circular dichroism spectroscopy of proteins: A practical approach for rapid acquisition of high-quality spectra

Circular dichroism (CD) is widely used to characterize proteins and assess the quality of biopharmaceuticals including proteins and nucleic acids. Rapid data acquisition is of great importance to broaden the range of CD applications. However, there are no consistent guidelines for selecting operating parameters such as bandwidth, digital integration time (DIT) and scan speed, so that an empirical rule of thumb approach is generally used. Here, we systematically investigated these parameters using rituximab as a model antibody. We found that a bandwidth of 2 nm, a scan speed of 50 nm/min, and a DIT of 4 s (far-UV) or 2 s (near-UV) preserved key spectral features while reducing noise and measurement time. Compared with conventional parameter values, this reduced the acquisition time for a CD spectrum to 1.4 min (far-UV) and 1.8 min (near-UV)-a 20- and ∼10-fold gain in efficiency, respectively. The secondary structure estimated from far-UV CD spectra remained consistent for all the parameter values used in this study, and was in agreement with the results of crystallographic analysis. Also, the acquisition time for single-wavelength thermal denaturation measurements was reduced from 70 to 18 min, and that for temperature-ramping spectra measurements from 270 to 90 min. These experiments allowed rapid evaluation of the stability of rituximab and revealed that aggregation-induced structural changes occurred immediately after denaturation.