Glycosylation analysis of therapeutic monoclonal antibodies

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.