| 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.
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.