Integrated Glycan Analysis of Monoclonal Antibodies Using UPLC-MS

This project at MassBiologics of University of Massachusetts Medical School will be led by Dr. David McNally and Dr. Tianyuan Peng. This project is part of a larger small group study to evaluate the variability associated with operator-associated variabil
Categories
Assays
Equipment and Supplies
Data

Industry Need

Biopharmaceutical manufacturers require accurate glycan analysis to ensure monoclonal antibody (mAb) quality, as glycosylation impacts efficacy, safety, and regulatory compliance. Current workflows often show variability in absolute quantitation due to complex sample preparation steps. Industry needs robust LC-MS methods that deliver consistent glycan profiles across analysts and days, reducing variability and improving confidence in process development and release testing.

Approach

UMass Medical School implemented an UPLC-MS glycan analysis workflow using the Waters BioAccord LC-MS system. Key actions:

  • Procured and installed BioAccord LC-MS instrument; developed SOPs for released glycan analysis (NISTmAb) and purified glycan standards (SRM 3655).
  • Applied Waters RapiFluor-MS kit for NISTmAb and 2-AB labeling for glycan standards.
  • Conducted inter-analyst and inter-day runs to assess precision and reproducibility.
  • Delivered comparative data on relative glycan abundance and absolute peak areas for NISTmAb and SRM 3655 samples.


Impacts

Upon project completion, the Waters BioAccord LC-MS system 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

UPLC-MS analysis achieved >96% mass confirmation of NISTmAb glycans and consistent relative abundance across analysts (e.g., G0F ~40.2% ±1.4%, G1Fa ~29.7% ±0.9%). However, absolute peak area CVs ranged 50–63%, indicating variance in quantitative recovery steps. This method supports reliable qualitative glycan analysis and highlights optimization needs for absolute quantitation.

Outputs/Deliverables

Installed and validated Waters BioAccord LC-MS system for glycan analysis.

Developed SOPs for released glycan and purified glycan assays.

Completed inter-analyst and inter-day precision study on NISTmAb and SRM 3655.

Generated datasets on relative glycan abundance (high consistency) and absolute peak areas.

Delivered recommendations for optimizing quantitative glycan analysis.

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Project Lead

University of Massachusetts System

University of Massachusetts System