Size Exclusion Chromatography Method Transfer of a Monoclonal Antibody Across UHPLC Systems

Significance of the Topic

The relative quantification of protein aggregates in monoclonal antibodies using size-exclusion chromatography (SEC) is vital for ensuring therapeutic safety and efficacy. Aggregates can induce immunogenic responses and reduce drug performance, so reproducible SEC analyses across different UHPLC platforms are essential.

Objectives and Overview of the Study

• Transfer an SEC method for monoclonal antibody analysis from an Agilent 1260 Infinity Bio-inert System to an ACQUITY Arc Bio System equipped with a 30 cm Single Zone Column Manager (CM-30S).
• Compare retention times, percent area of monomer, high molecular weight (HMW) species, low molecular weight (LMW) species, and repeatability between systems.
• Optimize column selection based on extra-column dispersion to maintain resolution.

Methodology and Instrumentation Used

Extra-column dispersion (5σec) was measured as 31 µL for the Agilent system and 50 µL for the ACQUITY Arc Bio System with CM-30S. A 7.8 mm × 300 mm SEC column packed with 3.5 µm particles (200 Å) was selected. The mobile phase consisted of 20 mM phosphate buffer with 350 mM sodium chloride at pH 6.8. Trastuzumab samples (10 mg/mL) were injected (10 µL) at 0.5 mL/min.

Used Instrumentation

• Agilent 1260 Infinity Bio-inert LC System
• ACQUITY Arc Bio System with Single Zone Column Manager (CM-30S)
• XBridge Protein BEH SEC Column (200 Å, 3.5 µm, 7.8 mm × 300 mm)
• Empower Chromatography Data System
• ACQUITY UPLC Tunable UV Detector

Main Results and Discussion

• Chromatograms on both platforms showed matched retention times and resolution for HMW, monomer, and LMW peaks, with a consistent non-resolved shoulder integrated into the monomer peak.
• Average resolution (Rs) between HMW and monomer and peak widths at 50 % and 4.4 % heights were equivalent across systems.
• Percent area results for HMW, monomer, and LMW species displayed no significant differences; repeatability (RSD) was ≤ 0.00 % for all analytes.
• Higher extra-column dispersion on the ACQUITY system was offset by appropriate column dimension selection, ensuring comparable separations.

Benefits and Practical Applications

• Enables robust transfer of SEC methods between UHPLC platforms without sacrificing quantification accuracy or chromatographic resolution.
• Supports legacy SEC methods with columns over 25 cm via the CM-30S, while offering expanded capacity for method development.
• Facilitates reliable monitoring of critical quality attributes such as aggregate levels in monoclonal antibody production and quality control.

Future Trends and Opportunities

Advanced column managers with greater multiplexing and automation capabilities can further reduce extra-column effects. Integration with high-resolution detectors and data analytics will enhance sensitivity and throughput. Combining SEC with mass spectrometry or multi-attribute methods holds promise for comprehensive biotherapeutic profiling.

Conclusion

The SEC method was successfully transferred from the Agilent 1260 Infinity Bio-inert System to the ACQUITY Arc Bio System with CM-30S, achieving matched performance in retention, resolution, percent area, and repeatability. The CM-30S enables compatibility with legacy column dimensions and expands capacity for future method development, ensuring reliable mAb aggregate analysis across platforms.

References

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3. Iraneta P., Koza S. High Resolution Size-Exclusion Chromatography Separations of mAb Aggregates, Monomers, and Fragments. Waters Application Note, 2020, 720006956EN.
4. Koza S., Reed C., Chen W. Impact of LC System Dispersion on SEC Analysis of Monoclonal IgG. Waters Application Note, 2019, 720006336EN.
5. Waters Spec Sheet, ACQUITY Arc System, 720005400EN.
6. Koza S., Reed C., Chen W. Evaluating Impact of LC System Dispersion on SEC Analysis of Proteins. Waters Application Note, 2019, 720006337EN.
7. Hong P., McConville P. Dwell Volume and Extra-Column Volume: Impact on Method Transfer. Waters White Paper, 2016, 720005723EN.
8. Koza S., Hong P., Fountain K.J. SEC UPLC Method Development for Trastuzumab Degradation Products. Waters Application Note, 2012, 720004416EN.