Evaluating the Performance of the ACQUITY Arc Bio UHPLC System for the Size-Exclusion Chromatography Analysis of a Monoclonal Antibody

Significance of the Topic

Size-exclusion chromatography is essential for evaluating protein therapeutics under native conditions. Monitoring high molecular weight aggregates and low molecular weight fragments of monoclonal antibodies ensures product safety and stability. Reliable separation and quantification support regulatory compliance and patient safety.

Objectives and Study Overview

This study compares three chromatographic systems for analyzing trastuzumab. It focuses on extra column dispersion and its impact on the resolution of aggregates and fragments. Systems include an HPLC platform, a biocompatible UHPLC system, and a high performance UHPLC system.

Methodology and Instrumentation

• Systems : Alliance HPLC, ACQUITY Arc Bio UHPLC, ACQUITY UPLC H-Class Bio
• Detectors : UV/Vis, tunable UV (TUV)
• Columns : XBridge Protein BEH SEC 200Å 3.5µm, ACQUITY UPLC Protein BEH SEC 200Å 1.7µm
• Software : Empower 3 for data acquisition and analysis
• Samples : Trastuzumab formulation, BEH200 protein standard mixture
• Mobile phases : Phosphate buffers with high salt concentration and water/acetonitrile mixtures

Main Results and Discussion

Extra column dispersion was lowest in the UPLC H-Class Bio system, intermediate in the Arc Bio system, and highest in the HPLC system. Lower dispersion translated into sharper peaks, higher plate counts, and better resolution between antibody monomer, aggregates, and fragments.

• High molecular weight aggregates were well resolved in all configurations.
• Low molecular weight fragments could only be quantified reliably with the 1.7µm UHPLC column or two 3.5µm columns in series.
• Tandem HPLC columns improved resolution but doubled analysis time and sample consumption.

Benefits and Practical Applications

• Enhanced aggregate and fragment detection supports quality control in biopharmaceutical production.
• Biocompatible UHPLC components resist corrosion in high salt buffers.
• System selection can be tailored to throughput, sensitivity, and resource constraints.

Future Trends and Possibilities

Advances in column materials and system design are expected to further reduce dispersion and analysis time. Integration with automated sample handling and real-time data processing will improve throughput. Emerging detector technologies may enhance sensitivity for trace-level impurities.

Conclusion

The biocompatible UHPLC system offers a balance between throughput and resolution for monoclonal antibody analysis. Minimizing extra column dispersion is critical for accurate quantification of aggregates and fragments. System and column choice must align with analytical goals and operational parameters.

References

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2. Bouvier ES Koza SM Advances in size exclusion separations of proteins and polymers by UHPLC TrAC Trends in Analytical Chemistry 2014 63 85 94
3. Neue UD HPLC columns Theory Technology and Practice 1997