High Resolution Ion Mobility Enables the Separation of α-Galactose Containing Glycans from Their Non-α-Galactose Isomers

Severe allergic reactions can occur when biotherapeutics containing the carbohydrate antigen galactose-α-1,3-galactose (α-Gal) are administered. The detection of α-Gal-containing N-glycans is a challenging task due to the presence of non-α-Gal-containing isomers. This study evaluates the ability of different analytical approaches to detect an α-Gal glycan in the presence of a non-α-Gal isomer. Cetuximab is known to contain N-glycans bearing the α-Gal epitope and was chosen for the study. N-glycans released from Cetuximab were analyzed using the HILIC-MS/MS system. HILIC-MS/MS successfully separated and identified α-Gal species when the Galactose number exceeded the antenna number. However, this system could not identify α-Gal species when the Galactose number equals the antenna number. A High-Resolution Ion Mobility Spectrometer (IMS) was also evaluated and found to resolve isomeric α-gal/non-α-gal isomeric glycans. The study demonstrates the utility of the IMS-MS system by detecting immunogenic glycans in the presence of their non-α-Gal isomers. To our knowledge, this is the first time this specific immunogenic pair of glycan isomers has been resolved in a biotherapeutic, which was enabled by a high-resolution IMS separation.