Biopharmaceutical manufacturers require accurate and reproducible glycan quantification for therapeutic antibodies because glycosylation impacts efficacy, safety, and regulatory compliance. Glycan synthesis is non-template-driven, making it highly sensitive to process variations. Current workflows often struggle with reproducibility and absolute quantitation. Industry needs robust LC-MS methods that ensure structural integrity and enable reliable glycan profiling to reduce risk and improve quality control.
Azadi Lab developed a procainamide-labeling LC-MS workflow for glycan analysis using:
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.
Evaluate whether “community performance metrics” can be defined for NISTmAb released and/or released/labeled methods
Understand if SRM 3655 can assist in harmonizing or standardizing glycan measurements
Evaluate modes of implementation of a new quantitative glycan standard, SRM 3655
Procainamide LC-MS analysis achieved consistent glycan profiles across analysts, with minimal differences in relative abundance for major glycoforms (e.g., G0F ~34–38%, G1F variants ~32%). Absolute quantitation of G0F via standard addition yielded 1.49 µg per mAb sample, confirming method accuracy. This approach supports reliable glycan QC and highlights opportunities for improved internal standards.
Developed and validated LC-MS workflow using procainamide labeling and instant PNGase F release.
Adapted Agilent AdvanceBio kit for cleanup and integrated fluorescence/MS detection.
Completed inter-analyst reproducibility study on NISTmAb and glycan standards.
Generated datasets on relative glycan abundance and absolute quantitation (G0F).
Delivered recommendations for improved internal standards and kit-based reproducibility.
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University of Georgia Research Foundation
