New Enzymatic Tools for O-Glycan Analysis Using LC-MS

摘要

Changes in protein glycosylation may impact the structure and function of a glycoprotein. For biopharmaceutical drug products, alterations in glycoprofile may affect efficacy and is therefore considered a critical quality attribute. Analysis of O-linked glycans have long suffered from lack of specific enzymes and there has been a need for new tools. A novel O-glycan specific endoprotease has been discovered in Akkermansia muciniphila (O-glycoprotease A, OgpA), digesting the protein backbone N-terminally of the glycosylated serine or threonine. In this study, the enzymatic acitivity of OgpA was utilized to develop analytical workflows and strategies for O-glycan characterization of biopharmaceuticals. Specific OgpA digestion and LC-MS/MS analysis were used to determine O-glycosylation site occupancy of etanercept, an Fc-fusion protein with a highly core 1 O-glycosylated hinge region, erythropoietin (EPO) with a single core 1 O-glycan and human chorionic gonadotropin beta chain (hCGp) with two N- and four O-glycans, of which one is core 2. To further facilitate the analysis of O-glycans, a sialidase mix from A. muciniphila has been characterized and developed, hydrolyzing a2-3, a2-6 and a2-8 linked sialic acids. Also, an O-glycosidase from Streptococcus oralis hydrolyzing de-sialylated O-glycans of core 1 and core 3 was used in the characterization workflows. The inherent site heterogeneity of etanercept O-glycosylation together with the OgpA specificity for O-glycan structures generated overlapping peptides that was used to map O-glycosylation sites in the hinge region of etanercept. After OgpA digestion of EPO, a specific digestion site N-terminally of the O-glycosylated serine(126) was detected using LC-MS. Digestion of hCGp was performed at native conditions with and without sialidase treatment and the RP-LC-MS result confirmed digestion also at core 2 type O-glycans. Taken together, strategies for analytical characterization of O-glycosylated proteins have been developed based on novel enzymes. The workflows enables site occupancy determination and composition analysis of O-glycosylated biopharmaceuticals and have applications for development of biologies and studies of structure-function relationships of glycoproteins.