Optimizing Mass Spectrometer Conditions to Accurately Determine Full/Empty AAV Ratios: A Sample Half-Full Approach

Significance of the Topic

Precise measurement of full versus empty adeno‐associated virus (AAV) capsids is crucial for quality control in gene therapy vector production. Overestimation or underestimation of filled capsids can lead to suboptimal dosing, reduced therapeutic efficacy, or unexpected safety concerns. Traditional methods like analytical ultracentrifugation require large sample volumes and lengthy analysis times. Emerging Orbitrap‐based charge detection mass spectrometry (CDMS) offers rapid, low‐volume analysis, potentially transforming AAV characterization workflows.

Objectives and Study Overview

This study aimed to identify which mass spectrometer parameters most strongly influence the accuracy and reproducibility of full/empty AAV ratios using CDMS. By mixing commercially available full and empty AAV2 capsids at known proportions (50 % and 75 % empty) and systematically varying instrument settings on a Q Exactive UHMR, the authors established optimal conditions for unbiased ratio determination.

Methodology and Instrumentation

Sample Preparation:

• Empty and full AAV2 capsids (2×10^13 vg/mL each) buffer‐exchanged into 200 mM ammonium acetate.
• Mixtures prepared at 50 % and 75 % empty capsid ratios.

Instrument and Operating Mode:

• Thermo Scientific Q Exactive UHMR Hybrid Quadrupole‐Orbitrap Mass Spectrometer.
• Direct Mass Technology mode for CDMS measurement.
• Electrospray ionization using borosilicate emitters at ~1.2 kV.
• Data processed with STORIboard software for mass determination and capsid classification.

Parameter Screening:
Baseline and variable ranges were defined for key parameters including ion transfer tube temperature, source DC offset, in‐source trapping (desolvation) voltage, injection flatapole RF, trapping gas pressure, HCD purge time, and field gradient. Each setting was adjusted individually to assess its effect on measured full/empty ratios.

Key Results and Discussion

Minimal Impact Parameters:

• HCD purge time and field gradient, trapping gas pressure, and extended trapping energy showed negligible bias on measured ratios.

Critical Biasing Parameters:

• In‐source trapping (desolvation) voltage: Increasing desolvation voltage caused progressive loss of full capsids, shifting measured ratios toward higher apparent emptiness.
• Injection flatapole RF voltage: Lower voltages reduced radial confinement of high m/z full capsids, exacerbating the observed bias when combined with elevated desolvation.

Optimization Strategy:

• Maintain moderate desolvation voltage while maximizing injection flatapole RF to retain full capsids.
• Adopt baseline settings (e.g., 350 °C ion transfer tube, –10 V in‐source trapping, 700 V flatapole RF) as starting point.

Benefits and Practical Applications of the Method

By applying optimized settings, CDMS enables:

• Accurate full/empty AAV ratio determination in under 10 minutes.
• Use of <1 µL sample volumes at therapeutically relevant titers.
• High reproducibility across replicate measurements.
• Applicability to multiple AAV serotypes with minor parameter adjustments.

These advantages support its integration into process development, release testing, and comparability studies for gene therapy vectors.

Future Trends and Opportunities

Advancements likely to further enhance CDMS for AAV analysis include:

• Improved signal processing algorithms to correct residual transmission bias.
• Automation of parameter optimization using machine learning.
• Exploration of alternative desolvation gases to balance ion retention and sensitivity.
• Integration with orthogonal techniques such as ion mobility to resolve partially filled capsids.

Such developments will expand throughput and robustness in viral vector characterization.

Conclusion

Systematic evaluation of instrument parameters on a Q Exactive UHMR revealed that in‐source trapping voltage and injection flatapole RF are the primary drivers of measurement bias for full versus empty AAV capsids. By adopting optimized baseline settings, the CDMS approach reliably quantifies full/empty ratios with minimal sample and time requirements, offering a powerful alternative to traditional AUC methods.

References

• Fort, K. L., van de Waterbeemd, M., Boll, D., Reinhardt‐Szyba, M., Belov, M. E., Sasaki, E., et al. Expanding the structural analysis capabilities on an Orbitrap‐based mass spectrometer for large macromolecular complexes. Analyst 143, 100–105 (2018).
• Goodwin, M. P., et al. White Paper 451: Addressing desolvation and ion transmission bias in high-mass CDMS measurements.