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Glycosylation analysis of therapeutic monoclonal antibodies


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Mauko, L 2011 , 'Glycosylation analysis of therapeutic monoclonal antibodies', PhD thesis, University of Tasmania.

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Pharmaceutical glycoproteins are one of the fastest growing therapeutic areas
and glycosylation plays an important role in biological activity of the drugs. Most
of the currently used therapeutic glycoproteins are expressed in non-human
systems, often producing glycosylation patterns which can reduce the
effectiveness and stability of the drugs or cause immune responses in patients.
Due to the structural complexity of glycans, glycosylation analysis remains
extremely challenging and frequently employs multiple separation and mass
spectrometry based approaches. The standard approach for glycan profiling
currently used in the pharmaceutical industry includes fluorescent labelling of
enzymatically-released glycans and separation with hydrophilic interaction
liquid chromatography (HILIC) coupled with fluorescence detection to achieve
appropriate chromatographic resolution, sensitivity and relative quantification.
In this study, new methods employing zwitterionic-type HILIC (ZIC-HILIC) for
the separation of 2-aminobenzamide (2-AB) labelled and reduced glycans from
therapeutic monoclonal antibodies were developed. By using the new approach,
the time of a chromatographic run was reduced compared to the standard
method and the new proposed methods were suitable for on-line coupling with
electrospray ionisation mass spectrometry detection (ESI-MS). Furthermore, it
was demonstrated that by using ZIC-HILIC, fluorescent labelling was not
required which significantly simplified the sample preparation.
To improve ESI-MS sensitivity of reduced glycans, the system was further
downscaled employing nano ZIC-HILIC, which enabled the detection of minor
glycan species. It was shown that coupling with high resolution MS provides
information on glycan composition; however, due to high numbers of possible
isobaric species, the confirmation of structures generally requires separation of
glycans in combination with exoglycosidase digestions and tandem MS based
approaches.The HILIC analytical approaches were additionally compared to an orthogonal
porous graphitized carbon (PGC) separation of reduced glycans coupled with
ESI-MS. PGC exhibited excellent capability for the separation of isobaric glycan
species and better sensitivity compared to HILIC. It was demonstrated that PGC
is highly suitable for analysis of extremely complex glycan samples and the
method was employed for structural characterisation of glycan species found in
monoclonal antibodies. Since the PGC ESI-MS method resulted mainly in
formation of protonated glycan species, the MS/MS spectra provided only
limited structural information. On the other hand, exoglycosidase digestions
enabled detailed characterisation of glycans from monoclonal antibodies. It was
shown that multiple analytical approaches are generally required to obtain a
complete glycan profile of a given glycoprotein.

Item Type: Thesis - PhD
Authors/Creators:Mauko, L
Keywords: LC-MS, glycosylation, monoclonal antibodies, HILIC, graphitized carbon
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