High Resolution and High Throughput Size-Exclusion Chromatography Separations of IgG Antibody Aggregates and Fragments on UHPLC and HPLC Systems with 2.5 μm BEH Particles

Importance of the topic

Size-exclusion chromatography (SEC) remains the standard technique for monitoring high-molecular-weight aggregates and low-molecular-weight fragments in monoclonal antibody (mAb) biotherapeutics. Accurate detection and quantification of these species are essential for product quality, stability assessment, and regulatory compliance in research, development, and quality control laboratories.

Objectives and Study Overview

• Compare Waters Protein BEH SEC 200 Å columns packed with 1.7 µm, 2.5 µm, and 3.5 µm particles on UPLC, UHPLC, and HPLC platforms.
• Evaluate separation efficiency, resolution (peak-to-valley ratio), plate counts, and throughput for IgG aggregates and fragments.
• Benchmark performance against two commercial sub-3 µm SEC columns.
• Provide practical guidance for selecting column particle size and internal diameter (I.D.) based on system dispersion and throughput requirements.

Methodology

A rituximab sample (~21 mg/mL) and BEH200 protein standards were analyzed on an ACQUITY UPLC H-Class Bio system with TUV detection at 280 nm. Columns of varying particle size (1.7, 2.5, 3.5 µm) and I.D. (4.6, 7.8 mm) were run with phosphate buffer (20 mM NaH₂PO₄, 400 mM NaCl, pH 7.2). Flow rates and injection volumes were scaled to column dimensions. System dispersion (5σec) was measured and controlled between 12.5 µL and 44.4 µL to assess extra-column effects. Resolution was quantified by peak-to-valley (P/V) ratios and efficiency by USP plate counts.

Instrumentation Used

• ACQUITY UPLC H-Class Bio chromatography system
• ACQUITY UPLC TUV detector (5 mm Ti flow cell, 280 nm)
• Empower 3 software
• Columns tested: Waters BEH SEC 200 Å (1.7, 2.5, 3.5 µm; 4.6×150–300 mm and 7.8×150–300 mm), Competitor A 2.7 µm 300 Å (7.8×300 mm), Competitor B 2.0 µm 250 Å (4.6×300 mm)

Main Results and Discussion

• Column efficiency increased with smaller particles; reduced plate heights were comparable across 1.7, 2.5, and 3.5 µm columns, confirming theoretical proportionality.
• On low-dispersion UPLC (5σec≈12.5 µL), the 1.7 µm, 4.6×300 mm column delivered highest HMWS-monomer (P/V up to 4.2) and LMWS1-monomer resolution, at the expense of higher backpressure and dispersion sensitivity.
• The 2.5 µm, 7.8×300 mm column matched 1.7 µm performance under optimal dispersion, produced consistent LMWS1 quantification (0.42–0.45% area) across 5σec up to 44 µL, and ran at ~33% longer times with lower pressure.
• High-throughput 150 mm formats showed the 1.7 µm column provided higher resolution at equivalent analysis times; the 2.5 µm, 7.8×150 mm format enabled comparable separations on UHPLC/HPLC with 50–75% longer runs.
• Compared to two commercial sub-3 µm columns, the 2.5 µm BEH SEC 200 Å column offered superior plate counts for IgG, BSA, and myoglobin, and unique separation of the ~100 kDa fragment due to optimized pore size.

Benefits and Practical Applications

• Tailored SEC assays for different LC platforms balancing resolution, robustness, throughput, and system dispersion tolerance.
• Reliable quantification of low-abundance fragments and aggregates in mAb development and QC workflows.
• Potential for high-throughput SEC or SEC-MALS for in-process monitoring (PAT) in biopharmaceutical manufacturing.

Future Trends and Potential Applications

• Integration of real-time SEC monitoring with process analytics in biomanufacturing.
• Further reduction of extra-column dispersion through optimized plumbing and LC hardware.
• Development of new hybrid particle chemistries for reduced non-specific interactions.
• Extension of optimized SEC methods to other protein therapeutics beyond IgG antibodies.

Conclusion

The 2.5 µm BEH SEC 200 Å column in a 7.8 mm I.D. configuration delivers robust high-resolution separations across UHPLC and HPLC systems with moderate run times and lower pressure sensitivity. For ultra-high efficiency on low-dispersion UPLC, the 1.7 µm, 4.6 mm I.D. column remains the top choice, albeit with higher backpressure and dispersion control requirements. The 3.5 µm format offers reduced pressure for broader instrument compatibility. These findings guide informed column selection based on system dispersion, desired throughput, and separation needs for mAb aggregate and fragment analysis.

References

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