Quantitative analysis of genome packaging in adeno-associated viruses using native MS

Importance of the Topic

Accurate determination of empty and genome‐filled adeno‐associated virus (AAV) capsids is essential for gene therapy development and manufacturing. Empty capsids can cause unwanted immune responses and skew dosage calculations, while precise quantification of genome packaging efficiency ensures consistent therapeutic potency. Conventional absorbance‐based assays often lack sensitivity and molecular specificity, highlighting the need for advanced analytical techniques.

Objectives and Study Overview

The main goal of this study was to evaluate the potential of intact native mass spectrometry (MS) on a Q Exactive UHMR hybrid quadrupole‐Orbitrap platform for rapid and accurate assessment of the empty:full capsid ratio in recombinant AAV8 preparations. The analysis focused on minimal sample handling, high sensitivity, and robust quantification across different mixture ratios.

Methodology

Sample preparation and analysis steps:

• Reference AAV8 material (2×10^13 particles/mL) was either diluted directly in 100 mM ammonium acetate (pH 6.8) or buffer‐exchanged via spin desalting columns to reach 1×10^11–1×10^13 vp/mL.
• Five microliters of sample were introduced using static nanoESI tips into the mass spectrometer without additional derivatization.
• Data acquisition employed positive‐ion mode with resolution settings up to 25 000 at m/z 200 and transient lengths tuned for rapid throughput.
• Signal clusters corresponding to empty and full capsids were integrated using image analysis software to calculate fractional abundance.

Instrumentation Used

• Mass spectrometer: Thermo Scientific™ Q Exactive™ UHMR hybrid quadrupole‐Orbitrap MS equipped with Nanospray Flex™ ion source.
• Ionization: Double‐coated borosilicate nanoESI emitters.
• Desalting: Thermo Scientific™ Zeba™ spin columns for buffer exchange.

Results and Discussion

Empty AAV8 capsids produced a signal cluster around m/z 24 000, whereas genome‐filled capsids appeared above m/z 28 000, reflecting an ~800 kDa mass shift corresponding to the therapeutic DNA. Mixed samples at 1:1 and 1:5 empty:full ratios yielded quantification results of approximately 58.5% and 76.6% full capsids, respectively. The assay proved robust across resolution settings from 3 500 to 25 000, consistently reporting ~61% full capsids in 1:1 mixtures. These findings confirm similar charge distributions for empty and full particles, enabling straightforward discrimination by mass difference.

Benefits and Practical Applications

• Minimal sample preparation reduces material loss and processing time.
• High‐throughput acquisition (<5 minutes per run) supports real‐time process monitoring.
• Direct mass‐based detection ensures unambiguous identification of capsid species.
• Quantitative accuracy aids dosage calculation and quality control in biomanufacturing.

Future Trends and Potential Applications

Integration of charge detection MS (CDMS) or charge‐reduction techniques could extend the assay to detect partially filled capsids. Expanding native MS workflows to other AAV serotypes and multiplexed assays will further streamline analytical pipelines for gene therapy vectors. Automated data analysis and coupling with upstream process analytics may enable closed‐loop manufacturing control.

Conclusion

This study demonstrates that intact native MS on the Q Exactive UHMR platform provides a rapid, sensitive, and accurate method for quantifying genome packaging in AAV capsids. Its minimal sample handling, robust performance across resolutions, and clear mass‐based discrimination of empty versus full particles make it a valuable tool for therapeutic vector characterization and quality assurance.

References

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