Detection and Quantitation of Succinimide in Intact Protein via Hydrazine Trapping and Chemical Derivatization

Abstract

Formation of aspartyl succinimide (Asu) is a common post-translational modification (PTM) 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, 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 imparted by fluorescence labeling include, the facile detection of the intact protein without proteolytic digestion to peptides; and high sensitivity, e.g. 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.

Deamidation and isomerization liability analysis of 131 clinical-stage antibodies

“…15,16 Additionally, at low pH, the Asu intermediate is relatively stable (maximally at pH 4-5) due to a reduced rate of hydrolysis of the 5-membered ring. [17][18][19] Under these acidic conditions, hydrolysis of the Asu intermediate becomes rate-limiting, permitting the accumulation of these species within the sample 20 and the subsequent facile detection of an 18 Da mass loss. This mass loss is detected and used as a surrogate for the isomerization modification, which is more difficult to detect via liquid chromatography-mass spectrometry.…”

Engineering an anti-CD52 antibody for enhanced deamidation stability

“…This methodology cannot differentiate between Asp and Iso-asp, but will approximate the amount of Asn conversion to Asp/IsoAsp. Due to the fact that deamidation may be induced by analytical method conditions, [38][39][40] we specifically monitored the amount of method-induced deamidation in our analyses. A time zero sample for each variant was used as a control to monitor deamidation resulting from the analysis.…”

Strategies for analysis of isomeric peptides

“…Recently, the Zhou group showed that if the sample treatment with protein isoaspartate Omethyltransferase is followed by treatment with 18 O-water, the methylated isoAsp intermediates will first cyclize and form a succinimide intermediate followed by hydrolyzation into isoAsp residues that are labeled by 18 O. As such, a 2 Da mass difference for each isoAsp residue is added to the peptide, and the site of isomerization can be determined directly with MS-fragmentation techniques such as CID [74][75][76][77][78].…”