Automated High-Throughput Permethylation for Glycosylation Analysis of Biologics Using MALDI-TOF-MS

Abstract

Monitoring glycoprotein therapeutics for changes in glycosylation throughout the drug's life cycle is vital, as glycans significantly modulate the stability, biological activity, serum half-life, safety, and immunogenicity. Biopharma companies are increasingly adopting Quality by Design (QbD) frameworks for measuring, optimizing, and controlling drug glycosylation. Permethylation of glycans prior to analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a valuable tool for glycan characterization and for screening of large numbers of samples in QbD drug realization. However, the existing protocols for manual permethylation and liquid-liquid extraction (LLE) steps are labor intensive and are thus not practical for high-throughput (HT) studies. Here we present a glycan permethylation protocol, based on 96-well microplates, that has been developed into a kit suitable for HT work. The workflow is largely automated using a liquid handling robot and includes N-glycan release, enrichment of N-glycans, permethylation, and LLE. The kit has been validated according to industry analytical performance guidelines and applied to characterize biopharmaceutical samples, including IgG4 monoclonal antibodies (mAbs) and recombinant human erythropoietin (rhEPO). The HT permethylation enabled glycan characterization and relative quantitation with minimal side reactions: the MALDI-TOF-MS profiles obtained were in good agreement with hydrophilic liquid interaction chromatography (HILIC) and ultrahigh performance liquid chromatography (UHPLC) data. Automated permethylation and extraction of 96 glycan samples was achieved in less than 5 h and automated data acquisition on MALDI-TOF-MS took on average less than 1 min per sample. This automated and HT glycan preparation and permethylation showed to be convenient, fast, and reliable and can be applied for drug glycan profiling and clinical glycan biomarker studies.

Determination of true ratios of different N-glycan structures in electrospray ionization mass spectrometry

“…MALDI-TOF MS of permethylated glycans circumvents the problems arising from carboxyl groups and is held to reflect truly the molar proportions of the glycans in a sample. This was convincingly substantiated in a recent study by comparison of MALDI-TOF MS and HILIC–HPLC data [36]. …”

Sialic acid linkage-specific permethylation for improved profiling of protein glycosylation by MALDI-TOF MS

“…More recently, automated permethylation and annotation of MALDI-TOF-MS spectra were introduced to structural glycomics [11–13]. Although permethylation combined with MALDI-TOF MS provides a good snapshot of the most likely glycan compositions, the exact structures of glycans in terms of the explicit linkages are difficult to assign, particularly those of sialylated glycans with higher molecular weights.…”

Recent Advances in the Mass Spectrometry Methods for Glycomics and Cancer

“…Shubhakar et al developed a high-throughput format to monitor glycosylation in biologics, however the same method can be applied to cancer tissues. 41 Derivatization of sialic acid to yield linkage information has been used by Holst et al to examine formalin-fixed tissue for MALDI imaging. 42 Specific stabilization and characterization of sialylated glycans was achieved with linkage-specific sialic acid derivatization employing ethyl esterification and dimethylamidation for differential analysis of α(2,3)- and α(2,6)-linked sialic acids in plasma and cancer tissues.…”