STABILITY CHARACTERIZATION OF MULTIPLE SEROTYPES OF ADENO-ASSOCIATED VIRUS USING CHARGE DETECTION MASS SPECTROMETRY

Significance of the Topic

Gene therapy applications depend on the integrity and stability of adeno-associated virus (AAV) vectors. Variations in capsid content (full, partially filled, empty) directly affect potency, safety, and dosing. Monitoring capsid composition under temperature stress is essential for reliable storage, transport, and quality control.

Objectives and Study Overview

This investigation aimed to evaluate the thermal stability of two AAV serotypes (AAV5 and AAV8) by quantifying capsid content distributions after incubation at 4 °C, 22 °C, 35 °C, and 50 °C. Charge detection mass spectrometry (CDMS) was employed to measure full, partially filled, and empty capsid abundances and derive key ratios.

Methodology and Instrumentation

A prototype benchtop CDMS system (modified Megadalton Solutions design) coupled to a Nanomate Triversa™ electrospray source was used. Calibration was performed using glutamate dehydrogenase standards.

• Sample Preparation: Full and empty AAV5 and AAV8 capsids (2.0 ×10¹³ vg/mL) were buffer-exchanged into 200 mM ammonium acetate with 0.01 % Pluronic™ F-68 and split into four temperature groups.
• Thermal Treatment: Aliquots were incubated for 30 minutes at 4 °C, 22 °C, 35 °C, or 50 °C without mixing, then analyzed directly in a 96-well plate.
• CDMS Analysis: Ions were trapped for 100 ms in the electrostatic linear ion trap, and mass, charge, and m/z data were collected until ~3000 ions per sample.
• Data Processing: Custom software binned mass and charge distributions to calculate average abundances and ratios of full, partial, and empty capsids.

Results and Discussion

AAV5: Full capsid abundance decreased by ~14 %, partial by ~10 %, while empty capsids increased by ~24 % from 22 °C to 50 °C.
AAV8: Full capsids declined by ~8 %, partial by ~10 %, and empty increased by ~18 % over the same range.
Full/empty, full/partial, and empty/partial ratios exhibited linear trends with temperature, indicating gradual loss of nucleic acid content rather than abrupt capsid collapse. Charge distribution shifts toward lower charge states suggest reduced structural integrity at elevated temperatures.

Practical Applications and Benefits

CDMS enables direct, label-free quantification of AAV capsid populations with minimal sample preparation. The approach supports rapid stability assessment, formulation screening, and quality control of gene therapy vectors.

Future Trends and Potential Applications

Integration of CDMS into biopharmaceutical QC workflows promises high-throughput serotype screening and real-time stability monitoring. Advances may include coupling with chromatographic separations, automation for multi-batch analysis, and extension to other large biomolecular assemblies.

Conclusion

Prototype benchtop CDMS successfully characterized thermal stability of AAV5 and AAV8, revealing ~10 % loss in full capsid content between 22 °C and 50 °C. The linear increase in empty capsids and charge shifts highlight gradual degradation processes. This method offers a robust tool for ensuring vector integrity in gene therapy development.

Reference

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