Detection and quantitation of succinimide in intact protein via hydrazine trapping and chemical derivatization

The formation of aspartyl succinimide is a common post-translational modification of protein pharmaceuticals under acidic conditions. We present a method to detect and quantitate succinimide in intact protein via hydrazine trapping and chemical derivatization. Succinimide, which is labile under typical analytical conditions, is first trapped with hydrazine to form stable hydrazide and can be directly analyzed by mass spectrometry. The resulting aspartyl hydrazide can be selectively derivatized by various tags, such as fluorescent rhodamine sulfonyl chloride that absorbs strongly in the visible region (570 nm). Our tagging strategy allows the labeled protein to be analyzed by orthogonal methods, including HPLC-UV-Vis, liquid chromatography mass spectrometry (LC-MS), and SDS-PAGE coupled with fluorescence imaging. A unique advantage of our method is that variants containing succinimide, after derivatization, can be readily resolved via either affinity enrichment or chromatographic separation. This allows further investigation of individual factors in a complex protein mixture that affect succinimide formation. Some additional advantages are imparted by fluorescence labeling including the facile detection of the intact protein without proteolytic digestion to peptides; and high sensitivity, for example, without optimization, 0.41% succinimide was readily detected. As such, our method should be useful for rapid screening, optimization of formulation conditions, and related processes relevant to protein pharmaceuticals.