Using DSC to investigate the impact of oxidation on protein structure

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00:00:00 Using DSC to investigate the impact of oxidation on protein structure
00:02:21 Using DSC to investigate the impact of oxidation on protein structure
00:03:07 Proteins form multiple levels of structure
00:04:46 DSC is a valuable tool to characterize conformational stability of proteins
00:06:02 Higher order structure characterization methods support two components of product characterization
00:07:51 Quality by Design: Product understanding & Process control
00:09:29 Applying HOS methods to QbD: Linking structural modifications to biological function
00:12:48 Using DSC to investigate the impact of oxidation on protein structure
00:13:34 DSC response is linear with respect to oxidation for many protein products
00:15:50 IgG2 has multiple structural domainsHeavy chain methionines are targets of oxidation
00:16:28 Primary structure: Formation of methionine sulfoxide
00:18:26 Secondary structure: No detectable change by FT-IR spectroscopy
00:19:27 Tertiary structure:No detectable change up to 86% oxidation
00:20:26 Size heterogeneity:No detectable change up to 86% oxidation
00:21:03 Thermal Stability Decreased with Oxidation: Shifts in Tm Detected by DSC
00:21:51 Thermal Stability Decreased with Oxidation: Shifts in Tm Detected by DSC
00:22:28 Potency comparable up to 39% oxidation
00:24:01 Summary of results
00:25:39 Oxidation, conformational stability, biological activity
00:27:58 Summary & Conclusions
00:29:36 Acknowledgements
00:30:20 Thank you for your attentionAny questions?
00:34:13 Contact Information
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.