Improved Size- Exclusion Chromatography of Adeno-Associated Viruses (AAVs) With XBridge Premier GTx BEH SEC 450 Å 2.5 μm Columns

Importance of Topic

Size-exclusion chromatography is a cornerstone technique for characterizing aggregation and size variants in biopharmaceuticals. In the rapidly evolving field of adeno-associated virus (AAV) gene therapies, accurate profiling of monomers, oligomers, and high molecular weight species is essential to ensure product safety, efficacy, and batch consistency. Advances in column hardware and particle technology address longstanding challenges of adsorption losses and limited resolution, enabling more robust quality control workflows.

Objectives and Study Overview

This study evaluates the performance of XBridge Premier GTx BEH SEC 450 Å columns packed with 2.5 µm particles and equipped with MaxPeak High Performance Surfaces for AAV aggregate analysis. Key aims include comparing kinetic efficiency with traditional 5 µm columns, assessing recovery of high molecular weight species under standard buffer conditions, demonstrating accelerated analysis times, and confirming column longevity using guard configurations.

Used Instrumentation

• UPLC System: ACQUITY H-Class Bio Plus with quaternary pump
• Detectors: Fluorescence (Ex 280 nm, Em 350 nm) and Tunable UV (230 and 260 nm) with titanium flow cell
• Columns: XBridge Premier GTx BEH SEC 450 Å 2.5 µm 4.6×150 mm analytical; matching 4.6×30 mm guard
• Vials: MaxPeak HPS treated screw neck vials

Methodology

AAV serotypes (AAV2, AAV5 empty/full, AAV9) at ~1×1013 vg/mL were injected (1 µL) without pre‐dilution, after optional centrifugation or filtration of aggregated stocks. The mobile phase comprised 10 mM phosphate buffer (pH 7.4) with 200 mM KCl, filtered and used within two days. Column temperature was maintained at 25 °C, sample tray at 6 °C, with typical flow rates of 0.25 mL/min for baseline comparisons and up to 0.6 mL/min for high-throughput trials.

Main Results and Discussion

• Particle Size Impact: Switching from 5 µm to 2.5 µm BEH particles increased plate count of the monomer peak from ~630 to ~2760, quadrupling separation efficiency without extending run times.
• Hardware Surface Chemistry: Hydrophilic HPS hardware minimized electrostatic adsorption, preserving recovery of high molecular weight species under moderate ionic strength. Conventional stainless-steel columns lost HMWS signals without extensive buffer optimization.
• High-Throughput Capability: At 0.6 mL/min the 2.5 µm column delivered full serotype profiles in ~5 minutes, matching aggregate quantitation (<15 % deviation) obtained with a 50-minute low-flow 5 µm method.
• Guard Column Utility: Adding a 30 mm guard extended column life and slightly improved resolution (Rs from 1.9 to 2.1) with minimal impact on analysis time, even after >40 AAV injections.

Benefits and Practical Applications

• Enhanced throughput for batch release and process development
• Improved quantitation of oligomeric and aggregated AAV forms
• Standardized method across serotypes using a single mobile phase
• Reduced method development and buffer screening efforts
• Extended column lifetime with guard protection

Future Trends and Opportunities

Further integration with multi-angle light scattering or mass spectrometry could deepen structural insights. Emerging sub-2 µm or core–shell particle formats may offer even higher efficiency. Automated sample preparation and inline bacterial monitoring could streamline high-throughput QC. Finally, standardized column chemistries and harmonized protocols across platforms will accelerate regulatory acceptance and method transfer.

Conclusion

XBridge Premier GTx BEH SEC 450 Å columns with 2.5 µm particles and MaxPeak HPS surfaces deliver superior kinetic performance, high recovery of high molecular weight AAV species, and drastically reduced run times. A single buffer system suffices for serotype-independent analysis, while guard columns enhance robustness and lifetime, making this approach well-suited for routine gene therapy quality control.

References

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