Robust Wide Pore Size Exclusion Columns for Virus-Like Particles (VLPs) Analysis

Significance of the Topic

Virus-like particles (VLPs) serve as noninfectious platforms in vaccine development and other biotherapeutic applications. Aggregation represents a critical quality attribute (CQA) that can impact product safety, efficacy, and manufacturability. Reliable quantification and resolution of monomeric and aggregated VLP species are therefore essential for process control and regulatory compliance.

Objectives and Overview

This study evaluates the performance of a newly developed Agilent AdvanceBio SEC column with 1,000 Å pore size and 2.7 μm particles for the analysis of recombinant human papilloma virus type 16 (HPV-16) VLPs. The goal is to demonstrate high-resolution separation of monomer, dimer, and higher-order aggregates, assess reproducibility across injections and column lots, and compare performance against a benchmark Bio SEC-5 column.

Methodology and Instrumentation

Reagents and Sample Preparation:

• HPV-16 VLPs at 1 mg/mL in 500 mM histidine, 100 mM NaCl, 0.02% Tween 80 (pH 6.0).
• Mobile phase: 50 mM phosphate buffer with 400 mM NaCl (pH 7.2), 0.2 μm filtered.
• Samples thawed on ice, centrifuged at low speed, injected in 1 μL volumes.

Instrumentation:

• Agilent 1290 Infinity II Bio LC system with binary pump.
• Detectors: diode array detector (DAD) at 220/280 nm and fluorescence detector (FLD) (excitation 280 nm, emission 348 nm).
• Column: Agilent AdvanceBio SEC 1000 Å, 4.6 × 300 mm, 2.7 μm.

Chromatographic Conditions:

• Flow rate: 0.35 mL/min at room temperature.
• Run time: 15 minutes per injection.
• Data processed with OpenLab CDS ChemStation software.

Main Results and Discussion

Detection Sensitivity:

• Ultraviolet detection at 220 nm provided stronger signal than at 280 nm.
• Fluorescence detection offered improved sensitivity for low-abundance species.
• Series connection of UV and FLD yielded comparable profiles, confirming UV sufficiency when sample quantity is ample.

Resolution and Separation:

• Baseline separation of monomer and dimer achieved with resolution ~1.6.
• Clear distinction of higher-order aggregates beyond the dimer peak.
• Retention time for VLP monomer around 9.0 minutes, in line with expectations from protein standards.

Reproducibility:

• Five consecutive injections exhibited 0.01% RSD in retention time and <2% RSD in peak area.
• Four different column lots showed <1% RSD for retention, monomer content, resolution, and tailing factor, confirming lot-to-lot consistency.

Comparison with Bio SEC-5 Column:

• AdvanceBio SEC column delivered sharper monomer peaks and improved resolution of aggregates due to optimized particle size and higher pore volume.
• Aggregate recovery and separation were enhanced relative to the benchmark column.

Benefits and Practical Applications

The AdvanceBio SEC 1000 Å column technology enables robust, high-resolution VLP aggregation profiling with minimal sample conditioning. High reproducibility supports quality control workflows in biomanufacturing. Dual-detection capability allows tailored sensitivity for diverse sample loads.

Future Trends and Opportunities

Integration with multi-angle light scattering or analytical ultracentrifugation can yield more comprehensive size and shape characterization. Advances in high-throughput SEC and automated sample handling will accelerate VLP screening. Emerging stationary phases may extend analysis to larger viral particles and complex conjugates.

Conclusion

The Agilent AdvanceBio SEC 1000 Å column demonstrates superior performance for the separation and quantification of HPV-16 VLP monomers, dimers, and aggregates. Its high resolution, sensitivity, and reproducibility make it a valuable tool for VLP CQA assessment and process optimization.

Reference

1. Schneider S. Determination of Protein Molecular Weight and Size Using the Agilent 1260 Infinity Multi-Detector Bio-SEC Solution with Advanced Light Scattering Detection. Agilent Technologies Application Note, 2014.