Comparison of Sub-3-μm HP-SEC Columns for the Analysis of IgG Antibody Aggregates (HMWS) and Fragments (LMWS)

Significance of the Topic

Size-exclusion chromatography (SEC) is essential for evaluating high molecular weight species (HMWS) and low molecular weight species (LMWS) in monoclonal antibody (mAb) biotherapeutics. Accurate aggregate and fragment profiling ensures product safety, efficacy, and regulatory compliance in biopharmaceutical development and quality control.

Objectives and Study Overview

This study compared the performance of the Waters BioResolve SEC mAb, 200 Å, 2.5 µm, 7.8 × 300 mm column with three commercially available sub-3 µm, 7.8 × 300 mm SEC columns for analyzing cetuximab aggregates (HMWS) and fragments (LMWS). Mobile phases optimized by each vendor were applied to minimize method bias. Key performance metrics included column efficiency, peak symmetry, and separation of monomer, dimer (HMWS1), multimer (HMWS2) and fragments (LMWS1, LMWS2).

Methodology and Instrumentation

• Sample preparation: Waters BEH200 SEC Protein Standard Mix and stressed cetuximab drug product (2 mg/mL).
• Chromatography conditions: columns 7.8 × 300 mm, flow rate 0.50 mL/min, 25 °C, injection volumes of 3 µL (standards) or 10 µL (cetuximab), duplicate analyses.
• Performance metrics: USP plate count (tangent method), reduced plate height, USP tailing at 5% height, USP half-height resolution, peak-to-valley ratio, and relative area of species.

Used Instrumentation

• Columns: Waters BioResolve SEC mAb, 200 Å, 2.5 µm, 7.8 × 300 mm; three commercial sub-3 µm, 7.8 × 300 mm SEC columns.
• LC system: ACQUITY UPLC H-Class Bio System (dispersion volume ~14 µL).
• Software: Empower 3 Chromatography Data Software.

Main Results and Discussion

• Column efficiency: BioResolve SEC mAb column exhibited highest USP plate count and favorable reduced plate height (h ~2.87) comparable to two other vendors (h = 2.81–3.04).
• Peak symmetry: BioResolve and one commercial column delivered near-ideal tailing factors (~1.0), whereas others showed slight tailing.
• Aggregate separation: All columns achieved similar monomer-dimer resolution. Larger pore columns provided enhanced multimer (HMWS2) separation, while smaller pore columns, including BioResolve, yielded stronger monomer-BSA resolution in standards.
• Fragment separation: BioResolve column and one vendor column achieved baseline separation of LMWS2 (50 kDa). Only BioResolve demonstrated a pronounced valley between monomer and LMWS1 (100 kDa) (P/V = 4.98 vs. 1.24), leading to accurate quantification of low-abundance fragments.

Benefits and Practical Applications of the Method

• Enhanced fragment resolution supports reliable detection and quantification of low-level cleavage products in mAb quality control.
• Robust performance across HPLC, UHPLC, and UPLC platforms simplifies method transfer and reduces dependency on specialized low-dispersion systems.
• Comparable aggregate analysis ensures consistent monitoring of product stability and safety within biopharmaceutical workflows.

Future Trends and Potential Applications

• Development of SEC columns with tailored pore sizes and particle chemistries to further improve fragment separation and throughput.
• Integration of online multiangle light scattering (MALS) or mass spectrometry for in-line molecular weight confirmation and structural characterization.
• Application to a broader range of protein therapeutics, including bispecific antibodies, antibody-drug conjugates, and fusion proteins.
• Advances in column packing and manufacturing to enhance robustness and extend column lifetime under high-throughput conditions.

Conclusion

The Waters BioResolve SEC mAb, 200 Å, 2.5 µm, 7.8 × 300 mm column delivers superior efficiency, symmetric peak shape, and robust fragment separation compared to other sub-3 µm SEC columns. Its optimized pore diameter and particle size enable reliable analysis of mAb aggregates and low-level fragments on diverse LC platforms, supporting stringent quality control and method transfer in biopharmaceutical laboratories.

References

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