DESIGNING A NEW PARTICLE TECHNOLOGY AND PH GRADIENT MOBILE PHASE CONCENTRATES FOR ROBUST, HIGH RESOLUTION CHARGE VARIANT ANALYSIS OF MABS

Significance of the Topic

Ion exchange chromatography (IEX) is a cornerstone technique for the characterization of monoclonal antibody (mAb) charge variants, which are critical quality attributes affecting safety and efficacy. Reliable separation of acidic and basic variants supports drug development, quality control and regulatory compliance.

Objectives and Overview of the Study

The study aimed to design a next-generation particle technology and develop a pH gradient mobile phase system to achieve robust, high-resolution charge variant analysis of mAbs across a wide range of isoelectric points (pI). Key goals included improving chromatographic resolution, column lifetime and method reproducibility.

Methodology and Instrumentation

The core innovation is a 3 µm non-porous polymeric sorbent grafted with sulfonic acid groups. Two stationary-phase prototypes (2 µm and 3 µm) were compared and the 3 µm phase selected for optimal pressure tolerance and stability. A mobile phase pH gradient was empirically optimized to balance resolution and run time.

Applied Instrumentation

• ACQUITY UPLC H-Class Bio system
• ACQUITY UPLC TUV detector
• Empower 3 chromatography software
• 4.6×50 mm BioResolve SCX mAb column (3 µm)

Results and Discussion

The BioResolve SCX mAb column with the tailored pH gradient demonstrated superior resolving power compared to competing technologies, especially for high-pI mAbs such as adalimumab and NIST mAb reference material. Lifetime testing showed consistent retention times, pressure profiles and resolution over 500 injections. Batch-to-batch reproducibility was confirmed across seven stationary-phase preparations, with RSD values below 2 %.

Benefits and Practical Applications

• High resolution of major and minor charge variants in a 20-minute gradient
• Robust column performance enabling elevated sample loads (up to 2 mg)
• Universal applicability across mAbs with pI 6–10
• Minimal method reoptimization when switching instruments or batches

Future Trends and Potential Applications

Further developments may include integration with multi-attribute monitoring workflows, automation of pH gradient generation, and expansion to other biotherapeutic formats such as fusion proteins or antibody-drug conjugates. Continuous improvements in particle engineering could push resolution and throughput even higher.

Conclusion

This work presents a novel IEX column technology and pH gradient system that collectively deliver robust, high-resolution charge variant analysis for mAbs. The approach combines enhanced particle design, reproducible manufacturing and finely tuned mobile phases to meet the demands of biopharmaceutical research and quality control.

References

1. Fekete S., Beck A., Veuthey J. L., Guillarme D. Ion-exchange chromatography for the characterization of biopharmaceuticals. Journal of Pharmaceutical and Biomedical Analysis. 2015, 113:43–55.
2. Auclair J. R., Rathore A., Krull I. Charge-Variant Profiling of Biopharmaceuticals. LCGC. 2018, 36(1).