Glycosylation is one of the most common protein post-translational modifications and can affect the protein’s functionality, stability and immunogenicity. It is for these reasons that for biopharmaceuticals there must be a full characterisation of any glycans to ensure their composition, pattern and location remains consistent to prevent any changes causing a loss of efficacy or unwanted side–effects.
During the production of a biopharmaceutical there are often many occasions where changes to the glycosylation pattern may occur, as the biopharmaceutical is produced in a living cell which can be affected by many differing parameters such as temperature, pH, availability of nutrients etc. All of these can cause a change in the glycosylation pattern and, as such, need to be fully characterised to meet regulatory demands. This article looks at six different strategies that can be employed to study glycans and offers a relevant example of each strategy.
Monosaccharide and Sialic Acid Analysis – If you wish to determine the structure and composition of your glycans then the individual monosaccharides and sialic acids must be identified and quantified. In this workflow the glycans are removed from the protein enzymatically using glactosidase and then broken down further to monosaccharides using acid hydrolysis. High performance anion-exchange chromatography, utilizing ion chromatography, with pulsed amperometric detection (HPAE-PAD) is then commonly used to analyse the monosaccharides and sialic acids. This workflow is very selective for carbohydrates, reproducible and simple to use. This technical note is a good example of the use of HPAE-PAD for monosaccharide analysis.
Labelled Glycan Analysis – Such a workflow is commonly employed to determine which glycan species are attached to the protein and their relative abundance. In this strategy the glycans are released from the protein; enzymatically for N-linked glycans and chemically for O-linked glycans. The glycans are then tagged with a fluorescent label, such as 2-aminobenzamide (2-AB), as they lack chromophores making detection difficult with LC-UV before separation and detection with HPLC and fluorescence detection. Such a workflow is described in this application note. A recent application note also describes a modified approach which simplifies and reduces sample preparation time for fluorescent labelling through the use of the Applied Biosystems™ GlycanAssure™ HyPerformance APTS kit. The application note can be read
High-Throughput Labelled Glycan Analysis – This workflow is similar to the one described above in that the glycans are released and fluorescently tagged, but instead of using (U)HPLC the workflow utilises capillary electrophoresis to achieve the analysis of up to 96 samples in about 9 hours. This strategy is achieved using the Applied Biosystems GlycanAssure workflow and more details can be gained from this
Unlabelled Glycan Analysis – This strategy is the same as the labelled glycan workflow, however, as the name suggests the glycans are not fluorescently tagged. Separation is still performed using (U)HPLC, but detection is performed using charged aerosol detection (CAD) which is a near-universal detector and suitable for analytes that do not contain chromophores, such as glycans. There are obvious benefits to this workflow in that by removing the labelling step you have a faster workflow with less steps which improves reproducibility. The UHPLC-CAD approach for analysis of unlabelled glycans can be seen in this application note.
Glycopeptide Mapping – Used predominantly to identify the site of glycan attachment on the protein as well as additional information on the glycan structure, the workflow steps involve digestion of the protein into peptides as in traditional peptide mapping approaches followed by liquid chromatography separation. The glycosylation site and structural information is then obtained with advanced LC-MS/MS fragmentation techniques such as electron transfer dissociation (ETD) and higher energy C-trap dissociation (HCD) using Thermo Scientific™ Orbitrap™-based mass spectrometry. Such a strategy is seen by some as an easier approach compared to released glycan workflows as there is no requirement to release, and in some instances label, glycans and attachment information is obtained. This application note demonstrates a fast and reproducible peptide mapping approach.
Intact Glycoform Analysis – This workflow is used for the rapid profiling of the pattern and degree of glycosylation. The intact biotherapeutic, in most cases a monoclonal antibody, is separated by liquid chromatography and then the glycoprotein characterised using high resolution/accurate mass (HR/AM) mass spectrometry. The advantages of using the intact workflow are that there is minimal sample preparation and analysis can be performed in a matter of minutes. This whitepaper discuss the use of Orbitrap-based HR/AM mass spectrometry for intact glycoform analysis.
In summary, there are many different strategies that can be employed to study glycans and which strategy or strategies you employ is dependent on the questions that need to answer, but also on many other parameters such as throughput, ease-of-use and access to instrumentation. Many of these workflows are complementary and it may necessitate the use of a selection of these strategies to obtain a complete glycan characterisation.