Accurate Quantification of N-glycolylneuraminic Acid in Therapeutic Proteins Using Supramolecular Mass Spectrometry

Practical applications of innovative host-guest systems are challenging because of unexpected guest competitors and/or subtle environmental differences. Herein, a supramolecular mass spectrometry (MS)-based method using a synthetic host cucurbit[7]uril (CB[7]) was developed for identifying and quantifying N-glycolylneuraminic acid (Neu5Gc) in therapeutic glycoproteins, which critically reduces drug efficacy. The development of a reliable derivatization-free analytical method for Neu5Gc is highly challenging because of the interference by the abundant N-acetylneuraminic acid (Neu5Ac). CB[7] recognized the subtle structural differences between Neu5Gc and Neu5Ac. Distinct host-guest interactions between CB[7] and the two sialic acids produced a highly linear relationship between the complexation and concentration proportions of the two sialic acids in MS. Furthermore, the developed method had sub-picomolar quantification limits and a wide range of applicability for diverse glycoproteins, demonstrating the potential utility of this method as a reliable assay of Neu5Gc in therapeutic glycoproteins.