Covalent Labeling Denaturation Mass Spectrometry for Sensitive Localized Higher Order Structure Comparisons.

Protein higher order structure (HOS) describes the three-dimensional folding arrangement of a given protein and plays critical roles in structure/function relationships. As such, it is a key product quality attribute that is monitored during biopharmaceutical development. Covalent labeling of surface residues, combined with mass spectrometry analysis, has increasingly played an important role in characterizing localized protein HOS. Since the label can potentially induce conformation changes, protocols generally use a small amount of label to ensure that the integrity of the protein HOS is not disturbed. The present study, however, describes a method that purposely uses high amounts of isobaric label (levels that induce denaturation) to enhance the sensitivity and resolution for detecting localized structural differences between two or more biological products. The method proved to be highly discriminative, detecting differences in HOS affecting as little as 2.5-5% of the molecular population, levels at which circular dichroism and nuclear magnetic resonance spectroscopy fingerprinting, both gold standard HOS techniques, were unable to adequately differentiate. The methodology was shown to have comparable sensitivity to differential scanning calorimetry for detecting HOS differences. In addition, the workflow presented herein can also quantify other product attributes such as post-translational modifications and site-specific glycosylation, using a single liquid chromatography-tandem mass spectrometry (LC-MS/MS) run with automated data analysis. We applied this technique to characterize a large (>90 kDa), multiply glycosylated therapeutic protein under different heat stress conditions and aggregation states.