Analysis of Charge Heterogeneity in Rituximab Innovator and Biosimilar Monoclonal Antibodies Using Salt-Gradient Cation Exchange Chromatography

Importance of the topic

Recombinant monoclonal antibodies (mAbs) represent a cornerstone of modern biotherapeutics, addressing diagnostic and treatment challenges across oncology, autoimmune disorders and beyond. Charge heterogeneity arising from post-translational modifications can critically influence mAb efficacy, stability and immunogenicity, making robust analytical characterization essential for quality control in both innovator and biosimilar development.

Objectives and Study Overview

This study aims to compare the charge variant profiles of the innovator rituximab and its biosimilar counterpart using a salt-gradient cation exchange chromatography (CEX) method. Key goals include establishing a reproducible separation protocol, quantifying acidic and basic variant distributions, and evaluating method precision across multiple replicates.

Methodology and Instrumentation

Ultrafine details about instrumentation and method:

• Cation Exchange Chromatography: Salt-gradient separation on an Agilent Bio MAb NP5 PEEK column (4.6×250 mm, 5 μm).
• Chromatographic system: Agilent 1260 Infinity Bio-inert Quaternary LC with bio-inert flow path to prevent metal interaction, diode array detection at 280 nm.
• Gradient conditions: Multistep gradient combining water, NaCl (850 mM), NaH2PO4 (41 mM) and Na2HPO4 (55 mM) over 20 minutes at 0.75 mL/min.
• Sample preparation: 1 mg/mL mAb in 10 mM phosphate buffer pH 7.5; optional carboxypeptidase B digestion to assess C-terminal lysine variants.

Key Results and Discussion

Overlay chromatograms revealed distinct charge variant patterns between innovator and biosimilar rituximab. The main peak retention time was highly reproducible (mean 11.44 min, RSD <0.1), with precision in peak area above 95%. Acidic variants accounted for approximately 3.5% in the innovator and below 1% in the biosimilar, while basic species represented 3.2% and nearly 70%, respectively. CPB treatment confirmed removal of C-terminal lysine residues as contributors to basic heterogeneity. These data demonstrate both qualitative and quantitative differences in charge microheterogeneity.

Benefits and Practical Applications

The salt-gradient CEX approach offers high resolution and reproducibility, supporting stringent QA/QC requirements for mAb therapeutics. Rapid method development is facilitated by software-driven buffer optimization, reducing analytical timelines. Quantitative charge variant profiling aids in biosimilarity assessment, stability studies and batch-to-batch consistency monitoring.

Future Trends and Opportunities

Emerging directions include integration of mass spectrometry for direct variant identification, automated high-throughput screening of buffer conditions and adoption of multi-dimensional workflows to resolve complex PTM profiles. Advances in column materials and bio-inert system designs will further enhance sensitivity and robustness in biopharmaceutical analytics.

Conclusion

This work establishes a robust, high-throughput salt-gradient CEX method for in-depth charge heterogeneity analysis of innovator and biosimilar rituximab. The procedure delivers excellent precision, clear resolution of variant species and practical applicability for regulatory compliance in biotherapeutic development.