A Comparative Study of the Intact Mass, Subunit Mass, and Released Glycans of Two Rituximab Biosimilars Using High-Resolution LC/MS

Significance of the Topic

Therapeutic monoclonal antibodies face patent expiry, leading to rapid biosimilar development. Regulatory bodies require demonstration of high similarity in critical quality attributes such as intact mass, subunit composition, and glycosylation. High-resolution LC/MS enables detailed characterization, reducing reliance on extensive in vivo studies.

Objectives and Study Overview

This study evaluates two rituximab biosimilars against the innovator by applying four complementary workflows: intact mAb analysis, reduced heavy and light chain subunits, IdeS-digested subunits (Fc/2 and F(ab')2), and released N-glycan profiling. The aim is to compare sensitivity, throughput, and suitability of each approach for biosimilar assessment.

Methodology

• Intact mAb: Direct injection without sample preparation.
• Reduced subunits: Disulfide reduction with DTT (4 M guanidine HCl, 56 °C for 60 min).
• IdeS digestion: Enzymatic cleavage (IdeS protease, 30 °C for 30 min in Tris-HCl) yielding Fc/2 and F(ab')2 fragments.
• Released glycans: Automated on AssayMAP Bravo using GlykoPrep Rapid N-Glycan kit, InstantPC labeling; total prep ~3 h.
• Chromatography: Agilent PLRP-S or AdvanceBio Glycan Mapping columns with appropriate gradients.
• Mass spectrometry: Agilent 6545XT AdvanceBio LC/Q-TOF in high-resolution mode; workflows use tailored mass ranges and source settings.

Used Instrumentation

• Agilent 1290 Infinity II LC system with High Speed Pump, Multisampler, Thermostat.
• Agilent 1260 Fluorescence Detector for released glycans.
• Agilent 6545XT AdvanceBio LC/Q-TOF system.
• Agilent AssayMAP Bravo platform.
• Agilent MassHunter BioConfirm 10.0 for deconvolution and glycan assignment.
• AdvanceBio Glycan Mapping and PLRP-S columns; GlykoPrep Rapid N-Glycan kit (InstantPC).

Key Results and Discussion

• Intact mAb analysis achieved sub-10 ppm mass error for major glycoforms. Biosimilar 1 closely matched the innovator; biosimilar 2 showed greater deviation and varied C-terminal lysine truncation.
• Reduced subunits uncovered low-abundance glycans (Man5, G0) important for clearance and immunogenicity.
• IdeS-digested Fc/2 fragments provided enhanced sensitivity, detecting minor glycoforms and lysine variants.
• Automated released glycan profiling in ≈3 h delivered full glycan maps. Biosimilar 2 exhibited higher G0 and Man5, lower G2F, and unique afucosylated species (H4N3, H4N4) absent in other samples.
• Sensitivity increases with sample preparation complexity: intact mAb < subunit analysis < released glycan profiling.

Benefits and Practical Applications

• Flexible workflows allow rapid identity testing or in-depth glycan profiling according to development stage.
• Automation reduces hands-on time, error and improves reproducibility.
• High-resolution LC/MS delivers precise mass and glycoform quantitation to support CQA monitoring and regulatory submissions.

Future Trends and Possibilities

• Real-time LC/MS integration for bioreactor monitoring and process control.
• Advanced bioinformatics and machine learning for glycosylation prediction and comparability.
• Novel fluorescent and mass tags to enhance glycan detection.
• Microfluidic and multiplex platforms for increased throughput and lower sample consumption.

Conclusion

Agilent high-resolution LC/MS workflows across intact, subunit and released glycan analyses provide comprehensive, complementary data for rituximab biosimilar characterization. Automated glycan preparation with AssayMAP Bravo accelerates profiling while ensuring deep glycoform coverage, supporting efficient biosimilarity assessments.

Reference

1. Kirchhoff CF et al. Biosimilars: Key regulatory considerations and similarity assessment tools. Biotechnol Bioeng 2017;114:2696–2705.
2. US FDA. Scientific Considerations in Demonstrating Biosimilarity to a Reference Product. Guidance for Industry 2015.
3. Chung CH et al. Cetuximab-Induced Anaphylaxis and IgE Specific for Galactose-α-1,3-Galactose. N Engl J Med 2008;358:1109–1117.
4. Tangvoranuntakul P et al. Human uptake and incorporation of an immunogenic nonhuman dietary sialic acid. PNAS 2003;100:12045–12050.
5. Agilent Technologies. A Comprehensive Approach for Monoclonal Antibody N-linked Glycan Analysis from Sample Preparation to Data Analysis. 5991-8550EN.
6. Hu Z et al. Carboxypeptidase D is the only enzyme responsible for antibody C-terminal lysine cleavage in CHO cells. Biotechnol Bioeng 2016;113:2100–2106.
7. Agilent Technologies. LC/MS/MS Peptide Mapping Comparison of Innovator and Biosimilars Rituximab. 5994-1495EN.
8. Goetze AM et al. High-mannose glycans on the Fc region of therapeutic IgG antibodies increase serum clearance in humans. Glycobiology 2011;21:949–959.
9. Agilent Technologies. Streamlined Workflows for N-Glycan Analysis of Biotherapeutics Using Agilent AdvanceBio Gly-X InstantPC and 2-AB Express Sample Preparation with LC/FLD/MS. 5994-1348EN.
10. Liu P et al. Subunit Mass Analysis for Monitoring Multiple Attributes of Monoclonal Antibodies. Rapid Commun Mass Spectrom 2019;33:31–40.