Automated Workflow for Monoclonal Antibody N-Linked Glycan Analysis

Significance of the Topic

Monoclonal antibody glycosylation plays a pivotal role in therapeutic efficacy, stability, and immunogenicity. Rapid and robust analysis of N-linked glycans is essential during biotherapeutic development, clone selection, process optimization, and quality control to ensure product consistency and safety.

Study Objectives and Overview

This work presents a fully automated workflow for the release, labeling, and cleanup of N-glycans from monoclonal antibodies using the Agilent AssayMAP Bravo platform combined with the GlykoPrep InstantPC kit. A total of 24 antibody samples were processed in parallel within approximately three hours. Glycan identification and relative quantitation were achieved using an Agilent 6545XT AdvanceBio LC/Q-TOF coupled to a 1290 Infinity II UPLC with fluorescence detection.

Methodology

• Immobilization and denaturation of mAb samples on RX cartridges via the AssayMAP Bravo.
• On-cartridge enzymatic release of N-glycans using PNGase F at 45 °C for 30 minutes.
• Instant labeling of released glycans on-plate with InstantPC dye.
• Cleanup of labeled glycans through CU cartridges to remove excess dye and reagents.
• HILIC separation on an AdvanceBio glycan mapping column using a 28-minute gradient.

Instrumentation

• Agilent AssayMAP Bravo liquid handler equipped with RX and CU cartridges.
• GlykoPrep InstantPC kit modules for digestion, labeling, and cleanup.
• Agilent 1290 Infinity II UPLC system with fluorescence detector (Ex 285 nm, Em 345 nm).
• Agilent 6545XT AdvanceBio LC/Q-TOF mass spectrometer with Dual Jet Stream source.

Key Results and Discussion

The control human IgG standard yielded 17 well-characterized glycans, while 22 unique glycan structures were annotated across all samples. The automated workflow demonstrated high reproducibility, with coefficient of variation values below 10% for most glycoforms. Fucosylated species (G0F, G1F, G2F) dominated the glycan profile, accounting for over 90% of total abundance. Comparative quantitation by MS and fluorescence detection showed excellent agreement, with differences below 5%, confirming the reliability of the InstantPC label and LC/MS approach for relative glycan profiling.

Benefits and Practical Applications

• High-throughput processing of up to 24 samples in under three hours with minimal hands-on time.
• Enhanced MS and fluorescence signals provided by the InstantPC label enable sensitive glycan detection.
• Consistent and reproducible glycan profiles support biopharmaceutical clone selection, cell line development, and quality control workflows.
• Automated sample preparation reduces manual variability and increases laboratory throughput.

Future Trends and Potential Applications

Future developments will focus on fully integrating next-generation Gly-X InstantPC workflows on automated platforms, expanding glycan coverage through advanced labeling chemistries, and applying rapid glycan profiling to biosimilar comparability studies, process monitoring, and personalized medicine approaches.

Conclusion

The combined Agilent AssayMAP Bravo and GlykoPrep InstantPC workflow delivers a streamlined, reproducible, and high-throughput solution for N-glycan analysis of monoclonal antibodies. When paired with Agilent LC/Q-TOF and fluorescence detection, this method provides accurate structural identification and quantitative profiling essential for biopharmaceutical research and quality assurance.

Reference

• Arnold et al. The Impact of Glycosylation on the Biological Function and Structure of Human Immunoglobulins. Annu. Rev. Immunol. 2007;25:21–50.
• Agilent Technologies. GlykoPrep-plus Rapid N-Glycan Sample Preparation with InstantPC Kit. Instruction Manual.
• Oscar et al. Analysis of Monoclonal Antibody N-glycans by Fluorescence Detection and Robust Mass Selective Detection. Agilent Technologies application note 5991-8071EN, 2017.
• Wong et al. A Comprehensive Approach for Monoclonal Antibody N-linked Glycan Analysis from Sample Preparation to Data Analysis. Agilent Technologies application note, 2017.
• Yan et al. Comparison of Common Fluorescent Labels for LC/MS Analysis of Released N-Linked Glycans. Agilent application note 5994-0942EN, 2019.
• Agilent Technologies. N-Glycan Analysis: Better Together. Brochure 5994-1647EN, 2020.
• Agilent Technologies. AdvanceBio Gly-X N-Glycan Prep with InstantPC Kit: Simplified Workflow for Rapid FLD/MS Glycan Analysis. Brochure 5994-0918EN, 2019.
• Yan and Jones. Streamlined Workflows for N-Glycan Analysis of Biotherapeutics Using Agilent AdvanceBio Gly-X InstantPC and 2-AB Express Sample Preparation with LC/FLD/MS. Application note 5994-1348EN, 2019.