Strategies for the profiling, characterisation and detailed structural analysis of N-linked oligosaccharides

Abstract

Many post-translational modifications, including glycosylation, are pivotal for the structural integrity, location and functional activity of glycoproteins. Sub-populations of proteins that are relocated or functionally changed by such modifications can change resting proteins into active ones, mediating specific effector functions, as in the case of monoclonal antibodies. To ensure safe and efficacious drugs it is essential to employ appropriate robust, quantitative analytical strategies that can (i) perform detailed glycan structural analysis, (ii) characterise specific subsets of glycans to assess known critical features of therapeutic activities (iii) rapidly profile glycan pools for at-line monitoring or high level batch to batch screening. Here we focus on these aspects of glycan analysis, showing how state-of-the-art technologies are required at all stages during the production of recombinant glycotherapeutics. These data can provide insights into processing pathways and suggest markers for intervention at critical control points in bioprocessing and also critical decision points in disease and drug monitoring in patients. Importantly, these tools are now enabling the first glycome/genome studies in large populations, allowing the integration of glycomics into other 'omics platforms in a systems biology context.

Comparison of orthogonal chromatographic and lectin-affinity microarray methods for glycan profiling of a therapeutic monoclonal antibody

“…HILIC-FD in HPLC format is not a "high resolution method", but considerable improvements in both separation time and peak resolution have been reported when switching to the finer particle sizes of UPLC [63,89]. In UPLC format, HILIC-FD is equal or perhaps slightly superior to HPAEC-PAD in separation efficiency of some structures, but requires the additional steps of labelling and cleanup with the attendant potential for introductions of bias.…”

“…However, there is a larger body of published research employing and supporting the 2-AB label, including a web-based glycan identification database centred upon relative retention times [96][97][98]. HILIC users are about evenly split, with strongly entrenched preferences for separation of 2-AA-labelled glycans on polymeric amine-based columns [64,94,[99][100][101], or for separation 2-AB-labelled glycans on silica-based (or silica-polymer hybrid) amide-based columns [15,63,86].…”

“…Both require substantial training and expertise, expensive systems, and significant time-consuming and costly reagent/sample preparations, including deglycosylation (and fluorophore labelling in the case of HILIC-FD). Robotics-based high-throughput adaptations of HILIC-FD with associated glycan identification databases and exoglycosidase digestion prediction tools have been reported [40,[60][61][62][63][96][97][98]111]. These developments certainly augment and expedite the process, but also necessitate commitments not possible for modest laboratories with limited resources.…”

“…The addition of orthogonal methods provides complementary and corroboratory information to clarify instances of inconclusive data. High-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and hydrophilic-interaction chromatography with fluorescence detection (HILIC-FD) are among the most widely used and informative methods for the determination of glycoprotein glycosylation, and are industry standards in glycan analysis [53,[55][56][57][58][59][60][61][62][63][64]. However, both HPAEC-PAD and HILIC-FD require removal of glycans from the underlying protein prior to analysis, involve multiple steps are overall rather time-consuming and relatively expensive, demand significant operator experience and expertise, and involve considerable data analysis.…”

Tissue-Specific Glycosylation at the Glycopeptide Level

“…It has also been reported that cellular localization and protein structure influence/determine protein-and site-specific N-glycosylation (23,24). It should be noted that intact glycopeptide studies usually only allow the determination of glycan compositions; the identity of the oligosaccharide units and their linkage may be obtained from released glycan studies (5,6).…”