Multi-Level Workflows for Quantitative N-Glycan and Sialic Acid Analysis of Biotherapeutics

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Event Overview: 

Biotherapeutics such as monoclonal antibodies (mAbs) are frequently glycosylated, and the structure of attached N-glycans can affect immunogenicity, pharmacokinetics and pharmacodynamics. This can make glycosylation a critical quality attribute (CQA), making characterization of N-glycans an essential part of the development process. We present complete N-glycan quantitation workflow solutions using liquid chromatography (LC) with fluorescence detection (FLD) or mass spectrometry (MS) for four levels of biotherapeutic analysis: intact mass, mAb subunits, glycopeptides, and released glycans. Released N-glycans are labeled with InstantPC (a glycan dye that provides high FLD and MS signal) or 2-AB (a traditionally-used label) prior to separation by hydrophilic interaction liquid chromatography (UHPLC-HILIC). Sialylation of biotherapeutics can also be important, and we present a plate-based streamlined workflow for quantitation of total sialic acid.

• Relative quantitation of mAb glycoforms at intact protein and mAb subunit level using the highly sensitive LC/Q-TOF system

• Use of HILIC column for glycopeptide separation and quantitative analysis by LC/MS

• Rapid and reproducible sample preparation for released N-glycans (1 hour for InstantPC, 2 hours for 2-AB)

• Analysis of InstantPC labeled mAb and Fc fusion protein N-glycans, including relative quantitation by LC/FLD and glycan structure assignment by LC/MS

• Quantitation of total sialic acid using a plate-based colorimetric assay with a broad range of detection

Key Learning Objectives:

• Importance of N-glycan analysis for biotherapeutic protein

• Multi-level LC/FLD/MS approaches to quantitate N-glycans and sialic acid content of biotherapeutics

• Choosing a fluorescent dye and analytical separation method for released N-glycans

Speakers: Dr. Aled Jones, Bioconsumables Marketing Manager, Agilent Technologies

Dr. David Wong, Sr. Applications Scientist, LC/MS Solution, MSD Division, Agilent Technologies

Time and Date: Europe & US Broadcast: Tuesday, Mar. 31, 2020 at 9am EDT | 2pm BST | 3pm CEST

US Broadcast: Tuesday, Mar. 31, 2020 at 2pm EDT | 1pm CDT | 11am PDT

Asia Pacific: Wednesday, Apr. 1, 2020 at 8:30am IST | 12pm JST | 2pm AEDT

On demand available after final airing Mar. 31, 2021.

Sponsor: Agilent Technnologies

Register free: http://www.chromatographyonline.com/lcgc_w/biotherapeutics