Fast and semi-automatic workflow to analyse monoclonal antibody glycopeptides


Glycoproteins are proteins decorated with chains of sugars. Many important proteins, especially antibodies, are glycoproteins. However, knowing exactly what sugars are attached to glycoproteins is difficult because of the complex analytical tools and data analysis that is needed. The increasing accessibility of simple-to-use mass spectrometers will make it easier to identify the sugars. We have therefore developed and applied a smart algorithm that semi-automatically elucidates these structures, reducing data processing time from hours to only 30 minutes. We hope our method will accelerate glycoprotein research and development, as well as simplify quality control for commercial glycoprotein products.


Societal Impact

Glycans attached to antibody-based drugs can significantly affect the drug’s clinical safety and performance. Therefore, regulatory authorities, drug manufacturers and developers must ensure that the glycans can be comprehensively identified to indicate consistency in quality between manufacturing lots. This is currently an operational challenge for liquid chromatography-mass spectrometry (LC-MS)—the industry standard—because the data analysis is complex and cumbersome. Our algorithm GlycopeptideGraphMS semi-automates the analysis of data collected from a Waters BioAccord LC-MS system - a simple-to-use and compact LC-MS system. Data processing time is reduced from hours to under 30 minutes without compromising accuracy and quality of the data. The workflow can be deployed in bioprocessing operations swiftly without the need for highly trained chemists and expensive infrastructure.


Technical Summary

Tryptic digested glycopeptides are injected into Waters BioAccord LC-MS system and its processed data are analysed using GlycopeptideGraphMS. GlycopeptideGraphMS is BTI’s proprietary software that applies graph theory to cluster glycopeptides that have similar retention times. This also helps to filter glycopeptides from sugar-free peptides and reduces background noise. By defining the composition of one reference node, GlycopeptideGraphMS can automatically assign glycan compositions to the remaining nodes. Our workflow has been demonstrated to be semi-automatic, faster and more efficient than released glycan manual assignment.


 2021_06 Front Chem_Zach Pang

Figure 1. BioAccord-GraphMS workflow that offers faster and reliable analysis of glycopeptides



Pang, K.T., Tay, S.J., Wan, C., Walsh, I., Choo, M.S., Yang, Y.S., Choo, A., Ho, Y.S. and Nguyen-Khuong, T., 2021. Semi-Automated Glycoproteomic Data Analysis of LC-MS Data Using GlycopeptideGraphMS in Process Development of Monoclonal Antibody Biologics. Frontiers in Chemistry, 9, p.355.