LC/MS-based Intact IgG and Released Glycan Analysis for Bioprocessing Applications

Area of Science:

• Biopharmaceutical Analysis
• Mass Spectrometry
• Glycomics

Background:

• Robust antibody glycan analysis is critical for biopharmaceutical development, manufacturing, and clinical biomarker research.
• Existing methods can be time-consuming and labor-intensive, necessitating more efficient platforms.

Purpose of the Study:

• To develop and describe a novel 96-well plate-based workflow for analyzing intact IgG antibodies and released N-glycans.
• To demonstrate the workflow's utility in cell culture monitoring and clonal selection.

Main Methods:

• A 96-well plate workflow incorporating Protein A purification, PNGaseF digestion, 2-AB labeling, and SPE clean-up.
• Analysis using an Orbitrap Fusion Mass Spectrometer and LC/MS on the Waters UNIFI platform.

Main Results:

• The plate-based workflow provides IgG glycan profiles consistent with non-plate based methods and commercial kits.
• The method significantly reduces hands-on time compared to traditional approaches.
• The workflow successfully identified major glycan structure changes and post-translational modifications (PTMs) like C-terminal lysine clipping and N-terminal pyroglutamic acid formation.

Conclusions:

• The developed 96-well plate workflow offers a robust, efficient, and sensitive platform for antibody glycan analysis.
• This method is applicable for monitoring cell culture, clonal selection, and characterizing PTMs in biopharmaceutical research.