Automated High-Throughput N-Glycan Labelling and LC-MS Analysis for Protein A Purified Monoclonal Antibodies

Importance of the Topic

N-linked glycosylation critically influences the safety and efficacy of biotherapeutic antibodies by affecting immunogenicity, clearance rates, and effector functions. Continuous monitoring of glycan structures is essential for drug development, biosimilar comparability, and regulatory compliance. High-throughput, robust analytical workflows accelerate biotherapeutic characterization and quality control.

Objectives and Study Overview

This study aims to extend the GlycoWorks RapiFluor-MS sample preparation method by integrating an automated diafiltration step for Protein A purified monoclonal antibodies (mAbs) and coupling the workflow to rapid LC-MS analysis. Key goals include:

• Enabling buffer exchange or concentration of mAb samples prepared in any buffer or concentration range
• Automating glycan release, labeling, cleanup, and cleanup on the Andrew pipetting robot
• Implementing a five-minute UPLC-MS method on the BioAccord system for high-throughput analysis
• Demonstrating reproducible glycan profiling across diverse sample conditions

Methodology

The protocol employs a two-step automated workflow. Step 1 uses diafiltration in a 96-well 10 K MWCO plate to exchange buffers and concentrate Protein A purified samples. Denaturation, PNGase F deglycosylation, and RapiFluor-MS labeling are then performed on the Andrew liquid handler. Step 2 executes HILIC SPE cleanup. A fast gradient on an amide UPLC column with ammonium formate and acetonitrile mobile phases separates labeled glycans. Data acquisition is conducted by electrospray positive mode on the BioAccord LC-MS system, and glycan assignment uses accurate mass screening against a targeted library.

Used Instrumentation

• Andrew pipetting robot with Extraction and Vacuum modules
• 96-well 10 K MWCO filter plate for automated diafiltration
• ACQUITY UPLC I-Class PLUS system with Glycan BEH amide column
• BioAccord LC-MS system with RDa detector
• waters_connect software for acquisition and data processing

Main Results and Discussion

The automated diafiltration workflow delivered glycan profiles closely matching those from the manual protocol, achieving an average recovery of 73 percent after buffer exchange and consistent relative abundances of major glycoforms FA2, FA2G1, and FA2G2. Application to mAbs purified from both buffer and cell culture media provided reproducible glycan fingerprints, with percent relative standard deviation below 15 percent for key glycoforms. The method also quantified low-abundance species such as high-mannose M5 and sialylated variants, confirming its suitability for critical quality attribute assessment.

Benefits and Practical Applications

• High-throughput processing of 48 samples in under seven hours including sample preparation and analysis
• Flexibility to handle any buffer composition and broad concentration range
• Reliable glycan profiling for bioreactor harvests, downstream fractions, and biosimilar comparability
• Reduced manual handling and improved laboratory productivity

Future Trends and Potential Applications

Automation in glycan analysis is expected to evolve with expanded multiplexing, real-time data processing, and enhanced informatics tools. Extensions to other glycoprotein classes and integration of orthogonal detection techniques will broaden application scope. Machine learning–based glycan pattern recognition may offer deeper insights into product heterogeneity and process control.

Conclusion

The automated diafiltration GlycoWorks RapiFluor-MS protocol combined with rapid LC-MS analysis establishes a versatile high-throughput platform for N-glycan profiling of Protein A purified monoclonal antibodies. This workflow delivers consistent recovery, fast turnaround, and robust glycan characterization across diverse sample conditions, streamlining critical quality attribute assessment and supporting efficient biotherapeutic development.

References

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6. Waters Corporation. GlycoWorks RapiFluor-MS N-Glycan Kit Manual. 2017.
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