title: Technical decision making with higher order structure data: utilization of differential scanning calorimetry to elucidate critical protein structural changes resulting from oxidation.
abstract: Differential scanning calorimetry (DSC) is a useful tool for monitoring 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. This Note is part of a series of industry case studies demonstrating the application of higher order structure data for technical decision making. For this study, 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 in 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 shown to be a leading indicator of decreased antigen binding suggesting a subtle conformation change may be underway that can be detected using 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. Therefore, by using MS, DSC, and relative potency methods in concert, the intricate relationship between a primary structural modification, changes in conformational stability, and functional impact can be elucidated.
authors.affiliation: Amgen, Analytical Sciences, Longmont, Colorado.
authors.forename: Kelly K
authors.initials: KK
authors.lastname: Arthur
authors.forename: Nikita
authors.initials: N
authors.lastname: Dinh
authors.forename: John P
authors.initials: JP
authors.lastname: Gabrielson
mesh_terms: D001688:Biological Products / Q000737:chemistry*; D002152:Calorimetry, Differential Scanning; D002626:Chemistry, Pharmaceutical; D003661:Decision Support Techniques; D055808:Drug Discovery / Q000379:methods*; D004355:Drug Stability; D016014:Linear Models; D013058:Mass Spectrometry; D008715:Methionine / Q000737:chemistry; D008956:Models, Chemical; D010084:Oxidation-Reduction; D011487:Protein Conformation; D055550:Protein Stability; D011506:Proteins / Q000737:chemistry*; D013329:Structure-Activity Relationship; D013678:Technology, Pharmaceutical / Q000379:methods*; D013696:Temperature
chemical_list: D001688:Biological Products; D011506:Proteins; D008715:Methionine
keywords: calorimetry (DSC); oxidation; protein structure; proteins; stability; structure activity relationship; structure property relationship