COMPLETE AUTOMATED WORKFLOW FOR COMPARING THE N-GLYCAN PROFILE OF INNOVATOR AND BIO-SIMILAR INFLIXIMAB SAMPLES

Significance of the topic

The N-glycan profile of therapeutic antibodies critically affects their efficacy, safety, and immunogenicity. Accurate and reproducible glycan characterization is therefore vital in the development and quality control of originator and biosimilar monoclonal antibodies.

Study objectives and overview

This study presents a seamless, end-to-end automated workflow for comparing the N-glycan profiles of innovator and biosimilar infliximab. The approach combines robotic sample handling with advanced LC–MS analysis and streamlined data reporting to facilitate rapid, high-confidence glycan comparability studies.

Methodology

Sample preparation follows the GlycoWorks RapiFluor-MS protocol on an Andrew Alliance pipetting robot equipped with automated vacuum control. Glycans are labeled, captured on μElution plates, and eluted under controlled vacuum cycles to ensure consistent recovery. Separation is achieved by HILIC on an ACQUITY Glycan BEH Amide column, and detection uses both fluorescence and time-of-flight mass analysis. MS fragmentation data are acquired concurrently for structural confirmation.

Used instrumentation

• Andrew Alliance pipetting robot with automated INHECO TEC temperature-vacuum controller and iBoot G2+ power switch
• Waters ACQUITY UPLC I-Class Plus system
• ACQUITY RDa Time-of-Flight mass detector (ESI positive mode)
• ACQUITY FLR fluorescence detector (Excitation 265 nm, Emission 425 nm)
• UNIFI Scientific Information System with Glycan Application Solution

Main results and discussion

System reproducibility was validated over 20 consecutive injections of a standard RFMS-labeled glycan mix, showing stable relative abundances across major glycoforms. Comparative analysis of two originator infliximab batches revealed highly similar N-glycan distributions, while the biosimilar sample displayed a distinct shift in sialylated glycoform levels. Simultaneous MS/MS fragmentation improved confidence in assigning closely eluting isobaric structures.

Benefits and practical applications

• Reduced hands-on time and user interactions via automated vacuum cycles during sample preparation
• Shortened overall protocol duration by overlapping pipetting and incubation steps
• Minimized risk of well drying and sample loss through precise vacuum control
• Seamless data processing and reporting with customizable UNIFI templates for regulatory documentation

Future trends and potential applications

Integration of this workflow with higher-throughput robotic platforms and advanced data analytics, including machine-learning algorithms, could further accelerate glycomics research. Expansion to other biotherapeutic classes and implementation of real-time quality monitoring in manufacturing are promising avenues.

Conclusion

The fully automated GlycoWorks RapiFluor-MS workflow on the Andrew Alliance platform, coupled with the BioAccord LC–MS system and UNIFI software, delivers robust, reproducible N-glycan profiling. This end-to-end solution streamlines comparability studies for innovator and biosimilar antibodies, enhancing analytical efficiency and confidence.

References

1. Zhang X, Reed CE, Shion H, et al. Released N-linked Glycan Analysis Using the BioAccord System. Waters Corporation; 2019.
2. Reed CE, Fournier J, Vamvoukas N, et al. Automated Preparation of MS-Sensitive Fluorescently Labeled N-Glycans with a Commercial Pipetting Robot. SLAS Technology. 2018;2472630318762384.