Aggregate Analysis of Biotherapeutics

Significance of the Topic

Size exclusion chromatography (SEC) is a cornerstone technique in biopharmaceutical analysis for quantifying protein aggregation and fragmentation. Aggregates formed during cell culture, purification, or formulation can impact drug efficacy and safety, making accurate aggregate quantitation critical for establishing critical quality attributes and ensuring consistent product performance.

Goals and Study Overview

The whitepaper reviews Agilent’s SEC column portfolio—AdvanceBio SEC (1.9 µm and 2.7 µm), Bio SEC-5 (2000 Å), and PROTEEMA—outlining their design, performance characteristics, and application ranges. The aim is to guide users in selecting the optimal column for peptides, monoclonal antibodies, ultralarge biotherapeutics (AAVs, VLPs, mRNA, LNPs), and polymers, while demonstrating improvements in resolution, throughput, and robustness.

Methodology and Instrumentation

SEC separates biomolecules based on hydrodynamic size using aqueous mobile phases and inert stationary phases to minimize secondary interactions.

• Columns: AdvanceBio SEC (120–1000 Å pores), Bio SEC-5 (2000 Å), PROTEEMA (100–1000 Å).
• Stationary phase: Hydrophilically coated silica or diol-based media to reduce nonspecific adsorption.
• Instruments and detectors: Agilent 1260/Infinity II/III LC systems; UV detectors (220–260 nm), fluorescence, light scattering, refractive index.
• Mobile phases: Phosphate buffers (pH 6.6–7.4) with controlled ionic strength; optional sodium chloride for ultralarge pore columns.

Key Results and Discussion

AdvanceBio SEC columns demonstrated:

• Long service life with consistent monomer/dimer resolution over 1 300 injections.
• Low secondary interactions for hydrophobic ADCs and native mAbs, yielding sharp peaks and high recovery.
• Superior performance versus competitor columns: improved peak shape, reduced tailing, higher monomer recovery.
• High-throughput capability: 4.6×150 mm columns enabled up to 20 injections per hour with comparable resolutions at flow rates up to 0.7 mL/min.
• Optimal pore-size selection: 500 Å and 1000 Å columns for ultralarge biomolecules, demonstrating enhanced resolution for AAV-9, HPV-16, VLPs, and oligonucleotide ladders up to 3 kb.

Benefits and Practical Applications

Accurate aggregate profiling supports:

• Process development and lot release testing of biotherapeutics.
• Characterization of novel modalities such as gene therapy vectors and lipid nanoparticles.
• Method transfer across labs and sites due to batch-to-batch column reproducibility.
• Rapid troubleshooting using preconfigured protein standards kits.

Future Trends and Opportunities

Emerging needs include ultra-high-throughput SEC, integration with mass spectrometry and multi-angle light scattering detectors, and tailored column chemistries for next-generation biologics. Advances in sub-2 µm and core-shell particles will further reduce run times and solvent consumption, while expanded pore-size ranges will accommodate ever-larger biotherapeutic assemblies.

Conclusion

Agilent’s comprehensive SEC column portfolio addresses applications from small peptides to multimegadalton assemblies, delivering robustness, high resolution, and method consistency. By selecting the appropriate pore size and particle technology, analytical and QC laboratories can achieve reliable aggregate quantitation and streamline biotherapeutic characterization workflows.

Reference

No external references were provided in the source document.