Application of microfluidic modulation spectroscopy for simultaneous structural and thermal stability analysis of...

The secondary structure of proteins is extremely important because it contains some of the most fundamental information about how a protein folds into its tertiary and quaternary structures. The most widely used biophysical tools for analyzing protein secondary structures, however, suffer from major limitations such as low resolution, poor reproducibility, and a narrow concentration range. This study utilized Microfluidic Modulation Spectroscopy (MMS), a novel automated protein structural characterization technique, to analyze the secondary structure of nine commercial monoclonal antibodies (mAbs) under various concentrations and buffer conditions. The results revealed that diluting the mAbs in formulation buffer did not affect the protein structure, whereas reconstituting the mAbs from formulation buffer to PBS altered the protein structure by a small but detectable margin. In addition, properties such as the antibody subtype and target seem to have little relation to the secondary structure of the protein, based on the 9 mAbs tested in this study. However, differences or changes in the secondary structure of antibodies led to discrepancies in their thermal stability and melting temperature. This study shows that MMS can detect the secondary structure of monoclonal antibodies reproducibly and reliably, based on which we can derive the factors affecting the determination of the secondary structure from the experimental results of small peak shifts in the spectrum.