Isomer Level Identification and Quantification of N-Linked Glycans by LC-SLIM-QTOF-MS

This project at North Carolina State University will be led by David C. Muddiman. 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 monoclona
Categories
Assays
Equipment and Supplies
Data

Industry Need

Biopharmaceutical companies require accurate and reproducible glycan analysis for monoclonal antibodies (mAbs) because glycosylation impacts product safety, efficacy, and regulatory compliance. Current LC-MS workflows often fail to resolve glycan isomers, which can lead to incomplete characterization and variability across labs. Industry needs advanced analytical methods that improve resolution, enable isomer-level identification, and support robust quantitation to reduce risk and accelerate development.

Approach

NC State implemented a state-of-the-art LC-SLIM-QTOF-MS workflow incorporating ion mobility mass spectrometry (IM-MS) for glycan analysis. Key innovations:

  • Used INLIGHT isotopic glycan tagging chemistry for quantitative analysis.
  • Applied ion mobility separation to distinguish structural isomers (e.g., G1a vs. G1b) that LC-MS alone cannot resolve.
  • Quantified G0F glycan in NISTmAb using three approaches:
    • Standard addition
    • Internal calibration (heavy/light isotopic tags)
    • External calibration (linear and quadratic models).
  • Detected 52 of 54 glycans listed in NIIMBL’s reference report and identified additional unlisted species.


Impacts

Upon project completion, the the Agilent/MobilIon LC-SLIM-QTOF-MS and GlycoHunter 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.

Evaluate whether “community performance metrics” can be defined for NISTmAb released and/or released/labeled methods

Evaluate modes of implementation of a new quantitative glycan standard, SRM 3655

Understand if SRM 3655 can assist in harmonizing or standardizing glycan measurements

Value Statement/Outcomes

Ion mobility-enabled LC-MS detected 52 of 54 expected glycans and resolved isomers (e.g., G1a vs. G1b) in milliseconds. G0F quantitation achieved precision within ±10% across three calibration strategies, supporting robust comparability. This approach could significantly reduce method variability and mitigate regulatory risk for glycan-critical therapeutics.

Outputs/Deliverables

Developed and validated LC-SLIM-QTOF-MS workflow for glycan analysis.

Implemented INLIGHT isotopic tagging for quantitative glycomics.

Generated inter-day replicate data for NIST SRM 3655 glycan standards and NISTmAb.

Quantified G0F glycan using three calibration strategies.

Delivered comprehensive dataset on glycan identification, isomer resolution, and quantitation performance.

Presentations

Enders, J. R., The Importance of High-Resolution Ion Mobility Mass Spectrometry to Accurately Read Back the Complex Language of Biology, Technology Networks Webinar, November 10, 2022. https://go.technologynetworks.com/the-importance-of-high-resolution

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Project Lead

North Carolina State University

North Carolina State University