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Variability in the measured glycan distribution of therapeutic antibodies

This project at Complex Carbohydrate Research Center, University of Georgia will be led by Dr. Parastoo Azadi.
Project status
100% Completed

Industry Need

  • Traditional mass spectrometry (MS)-based strategies lack the capability to distinguish the large number of coexisting isomeric glycans that are indistinguishable by mass alone. 
  • Since chromatography alone can sometimes result in co-eluting peaks for glycan isomers, positional isomers of simple fluorescently tagged glycans can be distinguished by MS/MS, but this technique is not able to differentiate between linkage isomers. 


This project is part of a larger small-group study to evaluate the variability associated with operator-associated variability in measuring released glycans from a monoclonal antibody sample. The University of Georgia plans to introduce ion mobility (IM) coupled to mass spectrometry (MS), which offers substantial improvement in glycan characterization based on the capability of IM to resolve isomeric glycans prior to MS measurements.  

This project will utilize a Waters Synapt system, including an Acquity UPLC I-Class Plus LC and an Acquity UPLC Fluorescence Detector. The IM technology available on the Waters Synapt system is an orthogonal separation technique that can separate isomeric ions by the differences in their collisional cross sections, which are dependent upon their geometries and in turn are based upon their bond connectivity. Therefore, linkage isomers that coelute in the chromatography have identical mass and fragmentation with MS/MS will be distinguished, quantified, and separated using IM. This approach will be used to measure and quantitatively evaluate the glycan profile of a reference monoclonal antibody using glycan standards as well as the glycan profile of a reference monoclonal antibody where the release was performed at a central laboratory. 

Project steps: 

  • Fluorescently tag released glycans and analyze the glycans by the Waters Synapt system.  
  • Use fluorescent tags that are based on the structure of procainamide, which includes an aromatic fluorophore and a tertiary amine functionality, using the Agilent InstantPC labeling and cleanup kits in order to sensitively detect and quantitate released glycans from antibodies.  
  • Separate the released and labeled glycans by HILIC chromatography, and the glycans will be identified by a combination of tandem MS (MS/MS) and IM techniques available on the Waters Synapt system. 

Outcomes and Impacts

Following completion of this project, the Waters Synapt system, including an Acquity UPLC I-Class Plus LC and Acquity fluorescence detector, will be available on a priority basis for NIIMBL-related technology and workforce activities, within any necessary constraints of the host institution, to be negotiated with NIIMBL.

Updates, Related Publications, and Deliverables

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Project Lead

University of Georgia Research Foundation

University of Georgia Research Foundation