Site-Specific Identification of α2,6 and/or α2,3 Sialyl-Linkage Isomers in CTLA4-Igs Using Nano-LC-Quadrupole-Orbitrap-MS/MS.

Cytotoxic T-lymphocyte-associated antigen-4 (CTLA4)-Ig is a fusion protein used to treat autoimmune diseases. In glycoproteins, sialic acids are linked to galactose via either α2,6 or α2,3 linkages, with the latter being more rigid and crucial for extending the serum half-life of biotherapeutics. However, site-specific identification of α2,6 and/or α2,3 linkages in glycoproteins remains a significant analytical challenge. In this study, we performed site-specific N-glycan analysis with sialyl-linkage isomers in a CTLA4-Ig mutant (CTLA4-IgMT) produced in Chinese hamster ovary (CHO) cells co-overexpressing N-acetylglucosaminyltransferase-IV and α2,6-sialyltransferase. Since CHO cells produce α2,3-linked glycoproteins, wild-type CTLA4-Ig (CTLA4-IgWT) was used as a control. Following Pronase digestion, N-glycopeptides were enriched and analyzed using nano-liquid chromatography-quadrupole-Orbitrap-tandem mass spectrometry. Retention time analysis revealed that α2,6-linked N-glycopeptides exhibit lower hydrophobicity than their α2,3-linked counterparts. Fragmentation analysis showed that fragment ion intensity ratios ([sialic acid]+/[N-acetylglucosamine]+) are lower for α2,6 linkages. To improve the accuracy, multiple linear regression was applied to adjust fragment ion intensities, deriving compensation coefficients for N-acetylglucosamine, galactose, and sialic acid. Using this approach, structural alterations in CTLA4-IgMT were identified, including 6 additionally generated, 11 partially converted, and 6 completely converted N-glycans, yielding an overall α2,6 linkage proportion of 56.7%. Notably, α2,6 linkages were identified at N76 and N108 in the CTLA4 region but were absent at N207 in the Fc region. This study is the first to determine α2,6 and/or α2,3 linkages at three N-glycosylation sites in CTLA4-Ig, providing a strategy for distinguishing sialyl-linkage isomers.