Snag spot-on adenovirus titer & characterization with Stunner

Significance of the Topic

Rapid and accurate quantification of adenoviral vectors is critical in gene therapy, vaccine development and oncolytic treatments. Conventional methods such as UV/Vis absorbance at 260 nm, qPCR, electron microscopy and AEX-HPLC are often time-consuming, labor-intensive and provide limited data per assay.

Objectives and Overview of the Study

This application note evaluates the Unchained Labs Stunner platform with the Adeno Quant software to measure adenovirus capsid titer, empty/full capsid ratio, protein and DNA content, particle size and aggregation in a single assay. Three lots of CsCl-purified Adenovirus 5 from different vendors were characterized to demonstrate accuracy, precision and sample-specific insights.

Methodology and Instrumentation

Samples: Three vendor lots of CsCl-purified AdV5 stored at –70 °C, thawed once and maintained at 4 °C.

Measurement Workflow:

• 2 µL sample loaded directly, no dilution or denaturation.
• Four DLS acquisitions of 5 s each to obtain size and scattering data.
• Full UV/Vis absorbance scan to deconvolute turbidity, protein and dsDNA contributions.
• Integrated Unmix algorithms correct for light scattering and calculate molar extinction coefficients based on viral protein sequences and genome size.

Instrumentation:

• Stunner platform combining UV/Vis spectroscopy, dynamic light scattering (DLS) and static light scattering (SLS).
• Adeno Quant software with built-in algorithms for capsid titer and empty/full ratio.
• Optional 21 CFR Part 11 compliance and full automation for 96-well throughput.

Main Results and Discussion

1. Turbidity Correction and A260:

• Stunner’s mathematical removal of scattering yields rapid A260 readings in seconds without SDS-heat denaturation.
• Eliminates variability from large particle turbidity and reduces assay time from 30 minutes to seconds.

2. Multi-attribute Analysis:

• Total capsid titer, full capsid titer, free/aggregated protein and DNA are reported together.
• DLS intensity distribution distinguishes intact capsids, aggregates and small impurities.

3. Vendor Comparisons:

• Vendor 1: High purity and consistency with average total capsid titer 4.2×10^12 cp/mL (%CV 4.9) and full titer 3.4×10^12 vg/mL (%CV 4.4); 81% full capsids; low aggregates.
• Vendor 2: Monodisperse particles (Z-average ~122.6 nm, PDI < 0.1) but elevated free DNA indicates host cell DNA contamination.
• Vendor 3: Balanced protein and DNA levels but strong aggregation detected, suggesting stability issues.

Benefits and Practical Applications

• Rapid at-line QC: minimal sample prep, no dyes or labels, full characterization in under one hour for 96 samples.
• High throughput and automatable workflow compatible with GMP environments.
• Comprehensive insight into viral purity, titer and stability to guide process decisions and KPI/CQA development.

Future Trends and Applications

Integration of multi-angle light scattering, expanded software analytics and AI-driven data interpretation will further enhance viral vector characterization. The approach can be extended to other viral platforms, biologics and nanoparticle-based therapeutics. Adoption as a routine PAT (Process Analytical Technology) tool promises tighter process control and accelerated product development.

Conclusion

The Stunner platform with Adeno Quant offers a one-stop, label-free solution for adenovirus vector characterization. By uniting UV/Vis, DLS and SLS in one assay, it delivers rapid, accurate and robust multi-attribute data to support vaccine, gene therapy and oncolytic virus production.

Reference

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