Glycan characterization of the NIST RM monoclonal antibody using a total analytical solution: From sample preparation to data analysis.
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Glycosylation is an important attribute of biopharmaceutical products to monitor from development through production. However, glycosylation analysis has traditionally been a time-consuming process with long sample preparation protocols and manual interpretation of the data. To address the challenges associated with glycan analysis, we developed a streamlined analytical solution that covers the entire process from sample preparation to data analysis. In this communication, we describe the complete analytical solution that begins with a simplified and fast N-linked glycan sample preparation protocol that can be completed in less than 1hr. The sample preparation includes labelling with RapiFluor-MS tag to improve both fluorescence (FLR) and mass spectral (MS) sensitivities. Following HILIC-UPLC/FLR/MS analyses, the data are processed and a library search based on glucose units has been included to expedite the task of structural assignment. We then applied this total analytical solution to characterize the glycosylation of the NIST Reference Material mAb 8761. For this glycoprotein, we confidently identified 35 N-linked glycans and all three major classes, high mannose, complex, and hybrid, were present. The majority of the glycans were neutral and fucosylated; glycans featuring N-glycolylneuraminic acid and those with two galactoses connected via an 1,3-linkage were also identified.
author list (cited authors)
Hilliard, M., Alley, W. R., McManus, C. A., Yu, Y. Q., Hallinan, S., Gebler, J., & Rudd, P. M.
complete list of authors
Hilliard, Mark||Alley, William R||McManus, Ciara A||Yu, Ying Qing||Hallinan, Sinead||Gebler, John||Rudd, Pauline M