Enhancing AAV8 Analysis With Automated ELIT-CDMS Sample Delivery

Significance of the topic

Adeno-associated virus (AAV) vectors are widely used in gene therapy and require rigorous characterization because final preparations are heterogeneous mixtures of empty, partially packaged, full and overfull capsids. High-resolution, single-particle mass analysis is essential to quantify genome packaging, determine intact capsid masses (megadalton scale) and detect trace contamination or carryover between samples. Integrating automated sample delivery with Charge Detection Mass Spectrometry (CDMS) expands throughput and reproducibility for laboratories that handle limited or high-value AAV material.

Objectives and study overview

This study evaluated a prototype nano-electrospray ionization (nESI) autoloader coupled to an Electrostatic Linear Ion Trap (ELIT)-based CDMS (Waters Xevo CDMS) for:

• Automated delivery and post-injection flushing of multiple AAV8 samples.
• Assessing injection-to-injection reproducibility across five days with alternating Full and Empty AAV8 capsid standards.
• Quantifying carryover and demonstrating the effectiveness of the autoloader wash protocol.

Methodology and instrumentation

Samples: Commercially sourced AAV8 capsid standards (Empty and Full CMV-GFP) at an initial concentration of 2 × 10^13 vp/mL were buffer exchanged into 200 mM ammonium acetate with 0.01% Pluronic F-68 and diluted to 5 × 10^12 vp/mL to stress-test carryover removal.

Acquisition and washes: A prototype nESI Autoloader fed a single nESI emitter per day; each injection acquired for 15 minutes. Between AAV injections a sequence of washes (A: deionized water; B: magic mix 1:1:1:1 H2O:IPA:MeOH:ACN; A; C: 200 mM AmAc + 0.01% F-68) was performed. Wash cycles completed in under one minute.

CDMS detection principle and processing: Ions in the ELIT induce charge on a detection cylinder while oscillating. Fast Fourier transform (FFT) of the induced periodic signal yields ion oscillation frequency (m/z), and signal amplitude gives charge (z). Multiplying m/z by z produces single-ion masses. Data were processed using the Waters_Connect CDMS Toolkit to produce relative capsid quantifications.

Used instrumentation

• Prototype nESI Autoloader coupled to Waters Xevo CDMS (ELIT-based).
• Single nano-ESI emitter (one emitter used per day across injections).
• Biospin P-6 size-exclusion columns (Bio-Rad) for buffer exchange.
• Waters_Connect CDMS Toolkit for data processing.

Main results and discussion

Reproducibility and population integrity:

• Empty AAV8: Average measured empty-capsid ratios across five days were ~97.5–98.3% (RSD 0.31%). Average measured mass ~3.64 MDa with mass error typically ~1.8–2.5% from theoretical.
• Full AAV8: Measured full-capsid ratios per day varied around 53–56% (RSD 2.34%). Average measured mass ~4.46 MDa with mass error ~1.1–1.3% from theoretical.
• Across injections the expected AAV8 mass distributions remained clean and well resolved with no anomalous intermediate masses, indicating negligible contamination or profile distortion.

Carryover performance:

• Alternating Full and Empty injections demonstrated very low carryover. Residual Full capsid levels detected in subsequent Empty injections were 0.07–0.13% (trace levels), evidencing the effectiveness of the autoloader wash routine and emitter flush design.
• The fluidic design of the autoloader/nano-ESI capillary uses backpressure to push prior sample material back toward waste, aiding fast emitter turnover and minimal cross-contamination.

Mass accuracy and gas-phase adduction:

• nESI-induced adduction was reported to be consistently below ~2.5% for AAV samples, resulting in acceptable mass accuracy for megadalton species and limited impact on relative quantification.

Benefits and practical applications

The combined autoloader + ELIT-CDMS workflow offers several practical advantages for AAV analysis and related large biomolecular assemblies:

• Improved throughput by automating repeated injections and rapid wash cycles, reducing manual handling time.
• Lower contamination and carryover risk, critical when assessing high-value clinical-grade material or scarce samples.
• Single-particle mass measurements provide direct, high-resolution insight into genome packaging states (empty/partial/full/overfull) without relying on deconvolution of ensemble spectra.
• Reproducible intact-mass determination for megadalton capsids supports research, process development and QC/assay comparability.

Future trends and potential applications

Anticipated developments that would further enhance CDMS-based AAV workflows include:

• Greater automation and integration with laboratory information management systems for routine QC pipelines.
• Higher-throughput emitter arrays or multiplexed injection systems to scale sample throughput while retaining single-particle resolution.
• Software advances for more automated particle classification (empty/partial/full/overfull) and improved statistical reporting for regulatory workflows.
• Continued improvements in mass accuracy and charge resolution to resolve closely spaced packaging states and identify post-translational modifications or adduct patterns.

Conclusion

Coupling a prototype nESI autoloader to ELIT-based CDMS enables robust, reproducible, high-resolution analysis of AAV8 capsid populations with minimal carryover and accurate intact-mass determination for megadalton species. The approach increases throughput and analytical integrity for AAV characterization, making it attractive for research, process development and quality control environments that require sensitive detection of heterogeneous packaging states and trace contamination.

References

1. RCSB Protein Data Bank. Entry 2QA0.
2. Waters Corporation. Poster 720009427EN (2026) — Enhancing AAV8 Analysis With Automated ELIT-CDMS Sample Delivery.