Measurements

Stability

Stability may be considered one of the most important structural characteristics to monitor during development and formulation since it directly relates to the function of a biotherapeutic. When the secondary conformation of a protein changes, the active sites could be affected, oftentimes detrimentally. Using the AQS³pro to monitor the stability of a protein over an experimental set of conditions is simple and intuitive. By tracking specific wave numbers that are associated with characteristic secondary structural features and calculating a “similarity score” or using weighted spectral difference, it is possible to observe if a protein changes its conformation which may indicate degradation, denaturation, or possibly aggregation.  

Analyzing samples using the AQS³pro powered by Microfluidic Modulation Spectroscopy (MMS) provides a level of sensitivity that is unrivaled. Enhanced second derivative results are generated from the Absolute Absorbance spectra and further converted to a Delta plot highlighting subtle changes in the secondary structure. This enables early action to inform decisions for upstream development modifications and downstream formulation workflow optimization.

Delta plot and stability analysis of 13 commercially sourced BSA samples compared to the control. Second derivative spectra for each biosimilar were subtracted from ref sample 4112305 to highlight spectra differences. Stability analysis tracks signals corresponding to anti-parallel beta-sheet, parallel beta-sheet, and alpha-helix for each sample. Application Note Biosimilar Structural comparison of Commercially Source Reference Standards by MMS Rapidly Detects Subtle but Critical Differences to Correctly Predict Activity for Use in an ELISA Product.

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