Carter K. Asef1, Lohra M. Young2, Benjamin E. Draper2, Casey E. Vantucci1, Martin F. Jarrold2, Mason Chilmonczyk1, and Suraj Dhungana1
1Andson Biotech, Atlanta, GA; 2Megadalton Solutions, Bloomington, IN
Introduction
The emergence of complex biotherapeutics—such as adeno-associated viruses (AAVs), mRNA, and large protein assemblies—demands advanced analytical tools to resolve their intricate structures. Charge detection mass spectrometry (CD-MS) is emerging as an essential tool for measuring mega-molecules, offering unmatched sensitivity and resolution. CD-MS measures both mass-to-charge (m/z) and the charge (z) of individual ions, enabling direct mass determination of biomolecules into and beyond the megadalton range. However, applying CD-MS to mega-molecules requires overcoming challenges in sample cleanup and generating sustained and robust nano-electrospray ionization (nanoESI).
A key step in CD-MS analysis is sample preparation to ensure accurate high resolution mass measurement. This often means buffer exchange—desalting and replacing non-volatile buffers with MS-compatible solvents. Without this, residual salts and buffer components suppress ionization and compromise data quality. Conventional offline methods like manual spin columns and traditional dialysis are slow, labor-intensive, and risk sample loss—especially for fragile, limited samples. Often multiple wash and spin cycles are needed to generate sufficiently desalted samples for CD-MS analysis. Longer offline cleanup steps significantly increase the chances of sample degradation and precipitation.
Stable and robust nanoESI spray is equally critical for CD-MS analysis. As this technique measures single ions, extended acquisition time is needed for signal accumulation and good ion statistics. Unstable spray leads to incomplete data and poor reproducibility.
This application note describes the integration of a rapid online buffer exchange system (DynaChip X1TM) with a CD-MS system for the analysis of AAV capsids.
Experimental Methods
AAV sample with titer of ~1×1013 vp/mL (cceAAV2-CB-GFP described in DOI: 10.1016/j.omtm.2024.101206) was used for CD-MS analysis (Indiana University school of medicine).
Instrument
All measurements were performed on an in-house built CD-MS instrument (Megadalton Solutions). Data were acquired at an ion energy of 130 eV/z with a charge uncertainty of approximately ±0.8 e for trapping events of 104.6 ms duration.
Offline Sample Preparation and Analysis
Buffer exchange was carried out using two Micro Bio-Spin P6 columns (Cat. No. 7326225, Bio-Rad) following the manufacturer’s protocol. Samples were exchanged into 200 mM of ammonium acetate (Cat. No. AM9070G and 10977015, ThermoFisher). Electrospray ionization was performed by direct infusion using a Triversa Nanomate (Advion) equipped with a 5 µm emitter.
Online Sample Preparation and Analysis
For online buffer exchange, samples were manually loaded into the DynaChip X1TM sample loop (2 µL), injected at a flow rate of 0.5 µL/min using a carrier solvent of 1:1 methanol:water, and buffer exchanged into 200 mM ammonium acetate by delivering it as the conditioning buffer at 50 mL/hr. Online nanoESI measurements were performed using a 20 µm emitter (Fossiliontech), enabling sustained nanoESI spray for extended acquisition times.
Results & Discussion
The DynaChipTM platform houses a biochemically inert flow-through microfluidic device that facilitates rapid online buffer exchange, desalting, and removal of interferents using principles of tangential flow filtration and microdialysis. The clean sample is directly introduced to the CD-MS system via a coupled nanoESI emitter. The total spray time was modulated by adjusting injection volume and/or sample flow rate.
Figure 1: Charge versus mass scatter plot for cceAAV2 obtained using the DynaChip X1TM–CD-MS online buffer exchange workflow (blue) or offline buffer exchange followed by CD-MS analysis (black).
The integration of the DynaChip X1™ online buffer exchange platform with Megadalton’s CD-MS was demonstrated to provide analytical performance equivalence to conventional multi-step offline buffer exchange workflows (Figure 1). Streamlining sample preparation and introduction into CD-MS into a single step reduced the sample analysis time and improved operational efficiency by 5X.
Figure 2: Mass distribution measured for cceAAV2 using DynaChip X1TM–CD-MS online buffer exchange workflow (blue) or offline buffer exchange followed by CD-MS analysis (black). Mass distribution shows empty capsid (3.7 MDa), capsid that have packaged a full genome (5.0 MDa), and capsid with partial genome (4.5 MDa).
Table 1: Summary of molecular mass information using DynaChip X1TM–CD-MS
(Obtained by CD-MS measurement of cceAAV2 particles using online buffer exchange DynaChip X1TM–CD-MS workflow or offline 2x spin column buffer exchange followed by CD-MS analysis. FWHM = Full With at Half Maximum)
The integrated system successfully resolved full, empty, and intermediate cceAAV2 capsid populations, providing precise assessment of vector packaging heterogeneity (Figure 2). FWHM for individual peaks showed excellent agreement for the online and multi-step offline approaches (Table 1) suggesting comparable sample cleanup. Agreement in the measured mass and intensity indicates structural integrity of sensitive cceAAV2 was well preserved throughout the online workflow, confirming that the approach is gentle enough to retain conformation of delicate mega-molecules.
A critical advantage of the online approach was the delivery of sustained nanoESI spray via the DynaChip X1™ platform, which is essential for CD-MS experiments requiring extended acquisition times to achieve accurate measurements of m/z and z. Continuous spray stability was achieved for 12 min with a single injection of sample (2 uL) and flowing at 0.5 uL/min. This effectively mitigated spray instability and disruptions that typically result in incomplete datasets, diminished sensitivity, and compromised reproducibility. This sustained spray capability ensured robust ion transmission and consistent single ion measurements, enabling reliable characterization of high molecular weight biomolecules under native conditions.
These findings validate the DynaChip X1™–CD-MS workflow as a technically sound and efficient alternative to labor-intensive offline methods. By minimizing manual handling and reducing sample loss, the workflow enhances throughput, reproducibility, and data fidelity, making it an attractive next-generation online sample preparation system for advanced characterization of viral vectors and other megadalton-scale biotherapeutics.
Conclusions
The DynaChip X1TM streamlines CD-MS workflows with online buffer exchange and long sustained spray provided by direct nanoESI integration.
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