Regeneron Pharmaceuticals, Inc.
Methods for Identification of Scrambled Disulfides in Biomolecules

Abstract:

Disclosed are methods for identification of one or more non-native disulfide bonds in a biomolecule (e.g, an antibody). In an example, a method includes performing a digestion of the biomolecule under non-reducing conditions to provide a sample comprising a plurality of biomolecule fragments, contacting the sample to a separation column, applying a first mobile phase gradient comprising trifluoroacetic acid (TFA) and a small molecule additive to the separation column, applying a second mobile phase gradient comprising TFA in acetonitrile (ACN) and a small molecule additive to the separation column, performing a partial reduction procedure on the eluted sample, applying the partially reduced eluted sample components to a mass spectrometer, and performing a mass spectrometric analysis on the partially reduced eluted sample components to identify the one or more non-native disulfide bonds in the biomolecule.