Advancement of microfluidic modulation spectroscopy as a highly sensitive protein characterization technique for the...

The higher order structure (HOS) of a protein is vital to its function and activity, making it a critical component in the development of protein-based therapeutics. Characterization of HOS can be performed using various biophysical techniques, but many of these methods have limitations such as low throughput, complicated workflow, narrow concentration range, and low sensitivity. Microfluidic Modulation Spectroscopy (MMS) is an emerging technology that addresses these limitations, offering high sensitivity and automated analysis for protein secondary structure. This study evaluates and compares the different well plate formats and scan modes of two MMS instruments. The newer Apollo system features a high throughput 96-well plate format and sweep scan mode that allows a 50% reduction in sample volume consumption and measurement time compared to the previous system. By measuring two proteins with drastically different secondary structures, the results demonstrated that the measurements were highly repeatable (>99% repeatability by area of overlap) regardless of the well plate formats or the scan modes. The limit of quantitation (LOQ) for determining structural impurity using the sweep scan mode was 3.2% and significantly better than that of FTIR at 23% from previous studies. This work highlighted the advancement of MMS as a highly sensitive technique to detect small changes of protein structures due to aggregation or misfolding.