High Resolution Size-Exclusion Chromatography Separations of mAb Aggregates, Monomers, and Fragments using BioResolve SEC mAb Columns on UPLC, UHPLC, and HPLC Chromatography Systems

Importance of Topic

Size‐exclusion chromatography (SEC) is a cornerstone technique for monitoring monoclonal antibody (mAb) quality by separating aggregates, monomers and fragments under non-denaturing conditions. Reliable quantification of high molecular weight species (HMWS) and low molecular weight species (LMWS) ensures safety, efficacy and consistency of biotherapeutic products.

Objectives and Overview

This application note evaluates Waters BioResolve SEC mAb Columns (200 Å, 2.5 µm) in four formats (4.6×150, 4.6×300, 7.8×150, 7.8×300 mm) across three liquid chromatography platforms (UPLC, UHPLC, HPLC) with differing extra-column dispersion. A NISTmAb-based size variant standard, supplemented with IdeS-derived fragments, was employed to certify column performance and guide column‐system matching.

Methodology and Instrumentation

System dispersion was quantified via 5σec volumes measured with caffeine injections on zero dead-volume unions. Chromatographic separations used 50 mM phosphate, pH 7.0, 200 mM KCl mobile phase at 0.2 mL/min (4.6 mm I.D.) or 0.575 mL/min (7.8 mm I.D.) and 35 °C column temperature. Platforms and dispersion values:

• ACQUITY UPLC H-Class Bio: 5σec = 10 µL (150 mm) / 13 µL (300 mm)
• ACQUITY Arc Bio: 5σec = 30 µL
• Alliance HPLC: 5σec = 49 µL

Detection at 280 nm (TUV/TUV-Vis) and data processed in Empower 3.

Main Results and Discussion

• 7.8 mm I.D., 300 mm length columns delivered consistent high resolution of aggregates, monomer and fragments on all platforms.
• 7.8×150 mm columns showed moderate resolution loss for Fab/c fragments on high-dispersion systems.
• 4.6×300 mm columns on UPLC retained near-equivalent separation performance; shorter 4.6×150 mm exhibited significant resolution loss on higher-dispersion systems.
• Aggregate (dimer–monomer) USP resolution (HH) remained stable for all but the 4.6×150 mm format; values exceeded 1.75 across conditions.
• Fragment separation quality assessed by peak-to-valley (p/v) ratio indicated robust LMWS1&2 resolution on the two 300 mm columns for UPLC and UHPLC; p/v fell below 2 on HPLC for 4.6×150 mm.
• Correlation analysis revealed that increased extra-column dispersion and dispersion peak tailing strongly degrade main peak efficiency (5σ plates) and fragment resolution.

Benefits and Practical Applications

• Out-of-the-box performance guaranteed by individual column testing with a mAb size variant standard.
• High resolution separations on UPLC, UHPLC and HPLC by selecting appropriate column I.D. and length.
• Optimized workflows for mAb QA/QC laboratories to reliably quantify aggregates and fragments.
• Reduced sample and solvent consumption with 4.6 mm I.D. columns when UPLC dispersion allows.

Future Trends and Opportunities

• Further reduction of system dispersion via minimized tubing length and internal diameter.
• Development of next-generation particles and column hardware to improve tailing factors.
• Expansion of standardized mAb reference materials for broader comparability.
• Integration of inline dispersion monitoring to ensure method fitness.

Conclusion

Waters BioResolve SEC mAb Columns deliver reproducible, high-resolution separations of mAb aggregates, monomers and fragments when matched to LC system dispersion. The 7.8 mm I.D., 300 mm configuration is the most robust across platforms. Column-specific performance testing with a mAb size variant standard enhances confidence in routine biotherapeutic characterization.

Reference

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