Virus molecular weight and empty/full capsid ratio measurements on a Q Exactive UHMR mass spectrometer using Direct Mass Technology mode

Significance of the topic

Adeno-associated virus (AAV) vectors are central to modern gene therapy development. Accurate, rapid, and low-consumption determination of the fraction of capsids that contain genome (full) versus those that are empty is critical for product quality control, dosing, and safety. Orbitrap-based charge detection mass spectrometry (CDMS), implemented as Direct Mass Technology mode on the Q Exactive UHMR, provides a rapid alternative to analytical ultracentrifugation (AUC) with far lower sample consumption and acquisition times on the order of minutes.

Objectives and study overview

This application note demonstrates measurement of AAV molecular weight and empty/full capsid ratios on the Thermo Scientific Q Exactive UHMR using Direct Mass Technology and STORIboard processing. The goals were to (1) define instrument and processing parameters that yield accurate and reproducible empty/full ratios, (2) characterize parameter-dependent biases (especially for high m/z species), and (3) recommend a practical parameter set for routine AAV assessment across serotypes.

Methodology and workflow

Samples: Empty and full AAV2, AAV8, and AAV9 capsids were obtained and mixed volumetrically to target 25/75, 50/50, and 75/25 empty/full compositions. Buffer exchange was to 200 mM ammonium acetate using a 100 kDa MWCO filter. Electrospray was produced with borosilicate emitters and the NanoSpray Flex source.

Acquisition: Data were acquired on the Q Exactive UHMR in Direct Mass Technology mode. Typical acquisitions required 1–2 minutes per run. Nitrogen was used as the trapping gas; heavier gases (xenon, SF6) were noted as optional when available.

Data processing: Raw files were processed with STORIboard (build 1.0.24204.1) to assign masses and classify capsids as empty, partial, full, or over‑filled. STORIboard parameters (e.g., R2 threshold, time thresholds, S/N) were optimized to report molecular weight distributions and percent populations.

Instrumental setup

Key instrument and STORIboard settings used and recommended in this study include:

• Ion transfer tube temperature: 350 °C
• Source DC offset: 0 V
• In-source trapping (desolvation): −10 V
• Injection flatapole: 4 V
• Interflatapole lens: 3 V
• Bent flatapole: 2 V
• Extended (HCD) trapping: 5 eV
• Trapping gas (approx.): 5.0 × 10−10 mbar
• HCD purge time: 15 ms
• HCD field gradient: 65

STORIboard processor parameters included an R2 threshold of 0.99, duration threshold 0.1, minimum time-of-death 0.2, minimum time-of-birth 0.1, S/N threshold 1, apply frequency correction = True, and an m/z tolerance of 50.

Main results and discussion

Parameter sensitivity: Most instrument parameters had minimal impact on measured empty/full ratios, but two parameters strongly biased results toward under-counting full (high m/z) capsids:

• In-source trapping (desolvation) voltage: Increasing desolvation voltages progressively biased the measurement toward higher apparent percent empty capsids, consistent with preferential loss of high m/z (full) ions due to radial ejection or incomplete confinement during energetic desolvation.
• Injection flatapole RF voltage: Higher RF improves radial confinement of high m/z ions and promotes better desolvation; reducing RF produced significant differences in the average ions detected per spectrum between empty and full populations, exacerbating bias when combined with high in‑source trapping.

Trade-offs and sensitivity: Some settings (trapping gas, source DC offset) had modest effect on ratio accuracy but influenced sensitivity (average ions per spectrum). The authors recommend source DC offset = 0 V for balanced sensitivity and accurate ratios.

Reproducibility: Using the optimized parameter set, measurements across AAV2, AAV8, and AAV9 and across volumetric mixes (25/50/75% empty) showed accurate recovery of expected empty fractions with good reproducibility. Cross-instrument testing of 50% AAV8 also demonstrated consistent ratio determination.

Processing advances: Improved signal processing algorithms that account for mass-shifting ions increase robustness when measuring heavy macromolecular ions and help compensate for incomplete desolvation effects.

Benefits and practical applications

This Direct Mass Technology approach delivers the following practical benefits:

• Rapid turnaround: minutes per acquisition versus longer AUC runs.
• Low sample consumption: orders of magnitude less material required than AUC.
• High specificity: direct mass measurement distinguishes empty, partially filled, full, and over-filled capsids.
• Actionable QC data: suitable for process development and lot release analytics in gene therapy workflows when properly validated.

Recommendations and caveats

To obtain accurate and reproducible empty/full ratios for AAV using Q Exactive UHMR Direct Mass Technology mode:

• Minimize bias by keeping in-source trapping (desolvation) low (recommended −10 V) and maintain adequate injection flatapole RF to confine high m/z ions.
• Prefer performing desolvation primarily in the HCD cell rather than aggressive in-source desolvation to avoid radial losses.
• Use source DC offset = 0 V for optimal sensitivity and balanced detection of empty and full capsids.
• Use the STORIboard processing parameters provided to consistently classify capsid populations; verify settings with known mixtures and serotypes.
• Consider heavier trapping gases where available to aid ion confinement for very large complexes, though nitrogen proved adequate in these experiments.

Future trends and applications

Expected directions and opportunities include:

• Further improvements in signal processing and charge-assignment algorithms to better handle mass-shifting and partially desolvated ions.
• Standardization of CDMS workflows and reference materials to support regulatory adoption for gene therapy QC.
• Expansion of Direct Mass Technology to other viral vectors, virus-like particles (VLPs), and heterogeneous macromolecular assemblies.
• Integration with automated sample handling and higher-throughput acquisition for manufacturing environments.

Conclusion

Direct Mass Technology on the Q Exactive UHMR, combined with STORIboard processing, enables rapid, low‑consumption, and reproducible measurement of AAV molecular weight distributions and empty/full capsid ratios. Accurate results require attention to a few critical parameters—chiefly in‑source trapping and injection flatapole RF—to avoid bias against high m/z species. The instrument and processing recommendations presented provide a practical basis for implementing CDMS-based AAV analytics in research and quality control settings.

References

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2. Werle AK, Powers TW, Zobel JF, et al. Comparison of analytical techniques to quantitate the capsid content of adeno-associated viral vectors. Molecular Therapy Methods & Clinical Development 2021;23:254–262.
3. Mietzsch M, Liu W, Ma K, et al. Production and characterization of an AAV1-VP3-only capsid: An analytical benchmark standard. Molecular Therapy Methods & Clinical Development 2023;29:460–472.
4. Fort KL, van de Waterbeemd M, Boll D, et al. Expanding the structural analysis capabilities on an Orbitrap-based mass spectrometer for large macromolecular complexes. Analyst 2018;143:100–105.
5. Goodwin MP, Grinfeld D, Yip P, et al. Improved signal processing for mass shifting ions in charge detection mass spectrometry. Journal of the American Society for Mass Spectrometry 2024;35:658–662.