ac6b01639_si_001.pdf (2.36 MB)
Automated High-Throughput Permethylation for Glycosylation Analysis of Biologics Using MALDI-TOF-MS
journal contribution
posted on 2016-08-01, 00:00 authored by Archana Shubhakar, Radoslaw P. Kozak, Karli R. Reiding, Louise Royle, Daniel I. R. Spencer, Daryl L. Fernandes, Manfred WuhrerMonitoring
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.