Comparison of therapeutic antibody originator and biosimilar glycosylation using an integrated glycan labeling solution

Significance of the topic

Glycosylation is a critical quality attribute for monoclonal antibodies affecting efficacy, safety and immunogenicity. Accurate monitoring of N-glycan profiles ensures biosimilar products match originator reference standards and comply with regulatory guidelines.

Objectives and overview of the study

This study evaluates the performance of an integrated glycan labeling solution combining the GlycanAssure HyPerformance APTS kit with the Vanquish Flex UHPLC system and Accucore Amide HILIC column to compare the N-glycosylation profiles of the infliximab originator (Remicade) and its biosimilar (Inflectra).

Methodology

• Sample preparation completed in under 1.5 hours: denaturation at 80°C, PNGaseF digestion at 50°C, APTS fluorescent labeling at 55°C and cleanup with magnetic beads.
• Parallel labeling of a dextran ladder for glucose unit calibration.
• HILIC separation using a 2.1×150 mm, 2.6 µm Accucore Amide HILIC column with a binary gradient of acetonitrile and 100 mM ammonium formate (pH 4.4).
• Fluorescence detection at 455 nm excitation and 500 nm emission with high sensitivity micro flow cell.
• Data acquisition and analysis with Chromeleon 7.2 software using a fifth-order polynomial fit for GU assignment.

Used instrumentation

• Thermo Scientific Vanquish Flex Binary UHPLC system with high-pressure mixing pump and SmartInject technology.
• Accucore 150 Amide HILIC column, 2.6 µm, 150 Å, 2.1×150 mm.
• Vanquish Fluorescence Detector with biocompatible 2 µL micro flow cell.
• Magnetic stand, heating shaking drybath, pH benchtop meter, autosampler and essential labware.

Key results and discussion

Both Remicade and Inflectra yielded 37 well-resolved N-glycan peaks. Retention time and peak area repeatability met stringent precision criteria (RSD <5.5%). Minor yet consistent differences in relative peak abundances reflected distinct manufacturing processes while remaining within regulatory expectations. The dextran ladder calibration enabled reliable assignment of glucose unit values for peak identification.

Benefits and practical applications

• Streamlined workflow reduces hands-on time and potential errors.
• High-precision UHPLC separation boosts confidence in glycan profiling.
• Applicable to a wide range of therapeutic monoclonal antibodies for biosimilarity assessment and quality control.

Future trends and potential applications

Complementary approaches such as O-glycan analysis and middle-down mass spectrometry may extend this workflow to more complex glycoproteins. Advances in automation and data analytics will further accelerate glycoprofiling in biopharmaceutical development.

Conclusion

The integrated GlycanAssure HyPerformance APTS kit with Vanquish Flex UHPLC and Accucore Amide HILIC column offers a robust, high-throughput platform for N-glycan analysis of monoclonal antibodies, supporting biosimilar comparability and regulatory compliance.

References

1. EMA approval of first mAb biosimilars Remsima and Inflectra.
2. ICH Q6B guidelines on biopharmaceutical quality attributes.
3. EMA guideline on similar biological medicinal products.
4. Thermo Fisher AN72443 application note on HILIC glycopeptide separation.
5. SmartInject technology for UHPLC precision (PO72275).
6. TN147: Dextran ladder GU calibration method.
7. EMA Inflectra assessment report.