Date recorded: January 19 2016
Duration: 35 minutes 20 seconds
Differential scanning calorimetry (DSC) is widely used to monitor the thermal stability of the molecular conformation of proteins. Here, we present an example of the sensitivity of DSC to changes in stability arising from a common chemical degradation pathway: oxidation.Six protein products from three structural classes were evaluated at multiple levels of oxidation. For each protein, the melting temperature (Tm) decreased linearly as a function of oxidation; however, differences in the rate of change of Tm, as well as differences in domain Tm stability were observed across and within structural classes. For one protein, analysis of the impact of oxidation on protein function was also performed. For this protein, DSC was a direct indicator of decreased antigen binding, suggesting a subtle conformation change that can be detected by DSC prior to any observable impact on product potency.
Detectable changes in oxidized methionine by mass spectrometry (MS) occurred at oxidation levels below those with a detectable conformational or functional impact. By using MS, DSC, and relative potency methods in concert, the intricate relationship between a primary structural modification, conformational stability, and functional impact can be elucidated.