ENHANCED DECLUSTERING AND CHARGE-STRIPPING ENABLES MASS DETERMINATION OF AAVS IN TOF MS

Significance of the Topic

Adeno-associated virus capsids are leading vehicles in gene therapy, offering targeted delivery of genetic material. Accurate mass measurement of these ~3.7 MDa complexes is vital for ensuring product consistency and safety during development and manufacturing. However, inherent heterogeneity in viral protein composition and surface adduction complicates conventional mass spectrometric analysis.

Objectives and Study Overview

This study aims to combine enhanced declustering and extensive charge reduction to resolve overlapping charge states in time-of-flight mass spectra of AAV particles. The approach targets clear deconvolution of mass distributions for both empty and genome-containing capsids, improving confidence in vector characterization.

Methodology

AAV samples (AAV5 and AAV8) were buffer-exchanged into ammonium acetate and infused via nano-electrospray ionization. Two complementary strategies were implemented: enhanced declustering using a modified StepWave ion guide applying high-frequency voltages to reduce noncovalent adducts, and charge stripping via an electron capture dissociation cell to lower average charge states and increase m/z spacing between peaks.

Instrumentation Employed

• Waters Q-cIMS-ToF mass spectrometer equipped with SELECT SERIES Cyclic IMS.
• Modified StepWave ion guide with parallel plate electrodes for declustering.
• ExD WK-150 electron capture dissociation cell (eMSion) for charge reduction.

Main Results and Discussion

Application of the enhanced declustering module revealed fine features spaced by ~50 m/z units within the broad envelope of AAV signals, indicating reduced adduction. Charge reduction through the ECD device shifted charge state distributions from m/z ~20,000 to ~90,000, enabling identification of individual charge states. Deconvolution using a modified MaxEnt algorithm yielded a primary mass of ~3.580 MDa for empty capsids. Modeling with a multinomial distribution based on the stochastic assembly of VP1, VP2, and VP3 subunits ([0.01:0.03:0.96]) aligned with deconvolved peaks, suggesting predominant capsid species of VP ratios [0:1:59] and [0:2:58].

Benefits and Practical Applications

• Enhanced spectral clarity allows precise mass assignment of high-MW viral particles.
• Improved workflow for rapid assessment of empty-to-full capsid ratios.
• Potential integration into quality control pipelines for gene therapy vector production.

Future Trends and Opportunities

Ongoing efforts will explore ECD fragmentation footprints to verify sequence integrity and further refine mass accuracy. Comparative studies with protein-level analysis are expected to validate assembly models. Integration with advanced deconvolution algorithms may enable routine characterization of increasingly complex virus-based therapeutics.

Conclusion

The dual strategy of enhanced declustering and charge stripping on a modified ToF platform successfully deconvolves heterogeneous AAV capsid populations, providing reliable mass measurements essential for gene therapy development and quality assurance.

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