Agilent AdvanceBio SEC 2.7 μm columns with 500 and 1000 Å pores

Importance of the Topic

Ultralarge biotherapeutics such as adeno-associated viruses (AAV), virus-like particles (VLP), oligonucleotides, and lipid nanoparticles (LNP) are at the forefront of cell and gene therapy, as well as vaccine development. Rigorous characterization of their size, aggregation state, and purity is essential for research, quality control, and regulatory approval. Size-exclusion chromatography (SEC) remains a key technique for these analyses but demands columns with appropriate pore sizes, minimal secondary interactions, high resolution, and robust performance to handle molecules exceeding 1 MDa.

Study Objectives and Overview

This study introduces Agilent’s AdvanceBio SEC columns packed with 2.7 µm particles and engineered with 500 Å and 1000 Å pores. The aim is to demonstrate their suitability for high-speed, high-resolution separation of ultralarge biomolecules, evaluating throughput, resolution, batch-to-batch reproducibility, column lifetime, and compatibility with advanced detection methods.

Methodology and Instrumentation

Chromatographic conditions for the 500 Å columns (4.6 × 150 mm and 4.6 × 300 mm) employed a mobile phase of 50 mM sodium phosphate with 400 mM NaCl (pH 7.2) at 0.35 mL/min, with fluorescence detection (λex 280 nm, λem 348 nm) on an Agilent 1290 Infinity II Bio LC System. Column lifetime and comparison tests used the Agilent 1260 Infinity II Bio LC System with UV detection at 220 nm. Light scattering detection was also evaluated to assess background noise and suitability for molecular weight determination.

Main Results and Discussion

• High Resolution and Speed: A 150 mm, 500 Å column delivered Rs ≈ 2.10 for AAV8 while halving analysis time compared to a 300 mm column (Rs ≈ 3.29).
• Minimized Secondary Interactions: Hydrophilic surface chemistry yielded sharp peaks and reliable size measurements across multiple AAV serotypes.
• Reproducibility: PEG/PEO calibration curves and AAV/VLP injections demonstrated overlapping retention profiles across three manufacturing batches, confirming pore uniformity.
• Ruggedness and Lifetime: Over 1,000 injections of uridine maintained column efficiency and stable backpressure, indicating extended service life.
• Detection Compatibility: Standard conditioning produced low background noise, enabling direct coupling with light scattering, fluorescence, or mass spectrometry without extensive flushing.
• Competitive Advantage: Compared to competitor SEC columns, AdvanceBio SEC 500 Å and 1000 Å exhibited superior resolution and reduced tailing for AAV-9 and HPV-16 samples.

Advantages and Practical Applications

• Enhanced throughput for discovery and QC workflows.
• Accurate aggregation analysis of ultralarge therapeutics.
• Cost savings through extended column lifetime and reduced replacements.
• Flexibility to accommodate diverse detection techniques.

Future Trends and Potential Applications

As biotherapeutics evolve, SEC columns may integrate with multidimensional platforms and inline detectors such as multi-angle light scattering and mass spectrometry for deeper characterization. Advancements in stationary phase chemistry and particle engineering will further improve resolution and selectivity for novel modalities like extracellular vesicles and synthetic nanoparticles. Updated column formats (e.g., narrower bores) and accelerated methods will meet growing demands for high-throughput screening in process development.

Conclusion

Agilent AdvanceBio SEC columns with 500 Å and 1000 Å pore sizes combine high-resolution separations, rapid analysis, robust performance, and versatile detection compatibility, making them ideal tools for ultralarge biotherapeutic characterization. These columns enhance confidence in aggregate analysis, streamline workflows, and deliver reproducible results across discovery, development, and quality control.

Reference

No formal literature citations were provided in the source text.