Charge Variant analysis of mAb Biosimilars by Nexera UHPLC with a Shim-pack Bio IEX Column

Importance of the topic

Monoclonal antibodies (mAbs) form the backbone of modern biotherapeutics, and the rise of biosimilars demands robust analytical tools to ensure product quality and consistency. Charge variant profiling addresses critical quality attributes related to post-translational modifications that can impact efficacy, safety and stability of mAb products.

Objectives and overview

This work compares two ion-exchange chromatography (IEX) approaches—salt-gradient and pH-gradient—using Shimadzu Nexera UHPLC and a Shim-pack Bio IEX column to separate and quantify charge variants of a bevacizumab biosimilar. The study evaluates resolution, run time, and repeatability across six consecutive injections.

Methodology and instrumentation

A 5 mg/mL bevacizumab biosimilar in 50 mmol/L Tris-HCl (pH 8.0) was directly injected (5 µL) on a Shim-pack Bio IEX SP-NP column (4.6 × 100 mm, 3 µm) at ambient temperature. Detection was by UV at 280 nm with a 0.6 mL/min flow rate. Salt-gradient mobile phases comprised water, 40 mmol/L NaH2PO4, 50 mmol/L Na2HPO4 and 1 mol/L NaCl. The pH-gradient method used 10 mmol/L phosphate buffer (pH 6.0) and 10 mmol/L bicarbonate buffer (pH 10.0).

Key results and discussion

Salt-gradient IEX achieved baseline separation of acidic, main and basic variants within 5 minutes. The main peak represented ~51% of total area, with acidic ~35% and basic ~14%. pH-gradient elution resolved variants in 12 minutes, yielding ~67% main, ~23% acidic and ~9% basic. Both methods demonstrated excellent repeatability (<1% RSD for retention times, <2% RSD for peak areas over six injections). The discrepancy in relative peak percentages reflects incomplete overlap of closely eluting species and differences in elution mechanism (Na+ displacement vs. isoelectric elution).

Benefits and practical applications

• High resolution and rapid throughput for charge variant monitoring in QC labs
• Generic applicability to a broad range of mAbs (pI 7–9)
• Robust performance with low variability supports method validation

Future trends and prospects

Emerging directions include integration with mass spectrometry for structural elucidation, development of mixed-mode IEX phases to improve selectivity, and automation of pH gradients for high-throughput workflows. Machine-learning–driven method optimization may further enhance speed and resolution.

Conclusion

Both salt-gradient and pH-gradient UHPLC-IEX methods deliver reliable, high-resolution charge variant analysis of bevacizumab biosimilars. Each approach offers distinct advantages—faster run times vs. simplified mobile phases—while maintaining excellent repeatability, making them suitable for routine biopharma QC.

References

1. Farnan D, Moreno GT. Multiproduct high-resolution monoclonal antibody charge variant separations by pH gradient ion-exchange chromatography. Anal Chem. 2009;81(21):8846–8857.