Acquisition of a ‘Xevo G2-XS QTof’ Mass Spectrometer and Measurement of Monoclonal Antibody Glycan Heterogeneity by Orthogonal Methodologies

The project “Acquisition of a ‘Xevo G2-XS QTof’ Mass Spectrometer and Measurement of Monoclonal Antibody Glycan Heterogeneity by Orthogonal Methodologies” at the University of Michigan, a member organization of NIPTE, will be led by Dr. Anna Schwendeman.
Categories
Assays
Equipment and Supplies
Data

Industry Need

Biopharmaceutical developers require accurate glycan profiling for monoclonal antibodies (mAbs) because glycosylation impacts product safety, efficacy, and regulatory compliance. Current workflows often show variability across users, instruments, and methods, and some labeling techniques (e.g., 2AB) lack sensitivity for low-abundance glycans. Industry needs robust, reproducible LC-MS methods and best-practice guidelines to reduce variability and improve confidence in glycan characterization for biologics development and regulatory submissions.

Approach

The Schwendeman Lab implemented LC-FLR-MS glycan analysis using:

  • Agilent GlyX 2AB Express kit for glycan release and labeling.
  • Analysis on Waters Xevo G2-XS QTof coupled with UPLC and fluorescence detection.
  • Triplicate runs across three separate prep/analysis days for NISTmAb, glycan standard mixes (A1, A2), and G0F SRM.
  • Used Waters UNIFI software for glycan identification and quantitation, leveraging dextran standards and MS confirmation for confidence.
  • Evaluated injection volume effects (1–4 µL) and optimized at 2 µL for sensitivity and peak integrity.
  • Highlighted limitations of 2AB labeling and recommended alternative rapid-labeling kits (e.g., RapiFluor-MS, InstantPC).

Impacts

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

Help evaluate modes of implementation of a new quantitative glycan standard, SRM 3655

Assist in understanding if SRM 3655 can assist in harmonizing or standardizing glycan measurements

Value Statement/Outcomes

LC-FLR-MS analysis identified major glycans on NISTmAb (e.g., G0F ~37.9% ±0.9%, G1F ~28.3% ±0.2%) with inter-day variability <1% for relative abundance, despite absolute area fluctuations. Low-abundance glycans (~2–3%) required manual MS confirmation, underscoring sensitivity limitations of 2AB labeling. This workflow supports qualitative glycan profiling and informs best-practice recommendations for biologics developers.

Outputs/Deliverables

Installed and validated Xevo G2-XS QTof LC-MS system for glycan analysis.

Developed SOP for released glycan analysis using 2AB labeling.

Completed inter-day reproducibility study on NISTmAb and glycan standards.

Generated datasets on relative glycan abundance and sensitivity limitations.

Delivered recommendations for alternative labeling methods and best-practice guidelines for glycan profiling.

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Project Lead

National Institute for Pharmaceutical Technology and Education, Inc (NIPTE)

National Institute for Pharmaceutical Technology and Education, Inc (NIPTE)