Designing a New Particle Technology for Robust Charge Variant Analysis of mAbs

Importance of the Topic

Ion exchange chromatography is a cornerstone technique for characterizing charge heterogeneity in monoclonal antibodies (mAbs). Robust separation of charge variants is essential for understanding critical quality attributes, guiding structure–function studies, and satisfying regulatory requirements in biopharmaceutical development and quality control.

Objectives and Study Overview

This study presents the design, optimization, and performance evaluation of a new strong cation exchange column specifically engineered for mAb charge variant analysis. The primary aim was to achieve superior resolution, reproducible performance, mechanical robustness, and high load capacity compared to existing technologies.

Methodology and Instrumentation

• Samples: NIST mAb (RM 8671), adalimumab, trastuzumab, infliximab at 2.5–5 mg/mL.
• System: ACQUITY UPLC H-Class Bio with Empower 3 Software.
• Stationary phases: non-porous polymeric particles (1.7 µm and 3.1 µm), modified for hydrophilicity and grafted with sulfonic acid groups.
• Chromatographic method: salt gradient using 20 mM MES buffer (pH 6–7) with NaCl elution, 4.6 × 50 mm columns, 0.96 mL/min, 30 °C, UV detection at 280 nm.
• Performance metrics: peak-to-valley ratio, USP half-height resolution, effective peak capacity, carryover, pressure profile, lifetime testing.

Main Results and Discussion

Optimization of particle diameter showed 3 µm non-porous particles offered improved resolution and significantly lower backpressure versus sub-2-µm media. Mechanical strength testing confirmed stability up to 10–21 kpsi for selected polymer compositions. Tuning surface hydrophilicity minimized hydrophobic secondary interactions while maintaining sharp peak shapes. Sulfonic acid ligand grafting was refined through a design of experiments approach to balance retention, resolution, and equilibration time. Comparative studies demonstrated higher resolving power and lower injection carryover versus leading competitor columns. Batch-to-batch and column-to-column reproducibility tests across seven stationary phase productions yielded RSD ≤ 3 % for retention times and ≤ 5 % for resolution metrics. Load capacity experiments on a 4.6 mm column showed linear response to at least 500 µg mAb with minimal resolution loss, supporting preparative applications. Lifetime testing over 500 injections confirmed stable retention, resolution, and backpressure.

Benefits and Practical Applications

• Enhanced resolution of mAb charge variants for critical quality attribute monitoring.
• Reproducible performance supporting method qualification and routine QC.
• Mechanical robustness compatible with UPLC, UHPLC, and HPLC platforms.
• High loading capacity enabling fraction collection for downstream structure–function assays.
• Extended column lifetime and low carryover reduce downtime and cost.

Future Trends and Potential Applications

Further miniaturization of non-porous particles appears to offer limited gains for large proteins. Future developments may focus on integration with multidimensional chromatography workflows, expansion to other biotherapeutic proteins, and high-throughput screening in process development.

Conclusion

The BioResolve SCX mAb column combines a 3 µm non-porous polymeric core with a finely tuned sulfonic acid surface to deliver superior resolution, reproducibility, load capacity, and robustness for monoclonal antibody charge variant analysis. Its performance advantages make it a valuable tool for research, development, and quality control in the biopharmaceutical industry.

References

• Auclair JR, Rathore A, Krull I. Charge-Variant Profiling of Biopharmaceuticals. LCGC 2018, 36(1).
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• Goyon A et al. Determination of Isoelectric Points and Relative Charge Variants of 23 Therapeutic Monoclonal Antibodies. J Chrom B 2017, 1065–1066, 119–128.