Characterization and quantification of succinimide using peptide mapping under low-pH conditions and hydrophobic interaction chromatography.

Characterization of asparagine deamidation and aspartic acid isomerization is an important aspect of biotherapeutic protein analysis due to the potential negative effect of these modifications on drug efficacy and stability. Succinimide has long been known to be an intermediate product of asparagine deamidation and aspartic acid isomerization, but despite the key role of succinimide in these reactions, its analysis remains challenging due to its instability. We have developed a paradigm in which two interlinked analytical methods are used to develop an optimized approach to analyze succinimide. In the first method, low-pH protein digestion is used for detailed characterization of succinimide with peptide mapping. At low pH, succinimide is stable and can be analyzed with accurate mass measurements and tandem mass spectrometry to confirm its identity and localize its modification site. These results are then used to establish a hydrophobic interaction chromatography (HIC)-based method that can be used for release and stability studies. In this method, unmodified protein, deamidated products, and succinimide are well separated and quantified. Good correlation was obtained between the data from low-pH protein digestion-based peptide mapping and the HIC-based method. Method qualification showed that the HIC-based method is robust, accurate, and precise and has excellent linearity.