To enhance the efficiency of size exclusion chromatography (SEC) step, the chromatographic column was judiciously selected. Virus-like particles possess various surface characteristics, including charged and hydrophobic regions, which can lead to nonspecific interactions with the stationary phase and often result in peak tailing. To mitigate these issues, we opted for a column with a hydrophilic coating to prevent such undesirable interactions. Additionally, a column with a small particle size (2.7 µm) was required to increase separation efficiency. Given that AAV has a compact structure and measures approximately 20-26 nm (200-260 Å) in size, we selected a column with a pore size of 500 Å. Ultimately, the selected chromatographic column has all the required features to enable a baseline separation of single AAV particles from their aggregates and lower molecular weight species. Importantly, the flow path of the chromatographic system can also be a source of undesired secondary interactions . We addressed this issue using a bioinert instrument with a metal free flow path. Our instrument equipped with both a 20-angle MALS and DAD detectors provided complementary information on AAV8 assemblies. MALS demonstrated superior sensitivity for detecting AAV8 dimers and higherorder-aggregates compared to UV absorbance (Figure ), consistent with its inherent sensitivity to macromolecular size and concentration. The peak eluting between 6 and 8 minutes (Fraction 2 [F2], Figures ) corresponds to AAV8 dimers . It exhibited enhanced MALS signal. The DAD facilitated the detection of low molecular weight species, which might correspond to truncations of VP proteins or ssDNA (Figure ) . The relative proportion of AAV8 dimers, calculated as a percentage of total AAV signal (see Methods and Table ), was quantified using peak area integration. Importantly, the DAD enables AAV detection at multiple wavelengths (280 nm and 260 nm, [Figure ). Notably, the DAD's capability for multi-wavelength detection (280 nm and 260 nm, Figure ) allowed us to assess the A260/A280 peak area ratio for both monomeric and dimeric AAV8 assemblies. Leveraging the principle that nucleic acids exhibit maximal absorbance at 260 nm and proteins at 280 nm , we used this ratio as an indicator of relative ssDNA to protein content. A260/280 values between 0.6-0.7 are indicative for empty AAVs, ratios between 1.3-1.4 represent filled capsids . For the monomeric assembly (F3), prepared as a 50/50 mixture of full and empty capsids, the A260/A280 ratio was measured at 1.16. The dimeric assembly (F2) exhibited a lower ratio of 0.86. This indicates that the dimeric fraction has a higher proportion of empty capsids than the monomeric fraction. It is important to note that using the 260/280 ratio for quantitative analysis requires careful calibration of well characterized standards relevant to the AAV product being measured. An intrinsic limitation of the method is that coeluting partial filled or overfilled AAVs are not accounted for. Thus, to validate our analytical workflow, we determined the AAV8 content in the sample mixture using CD-MS. Our chromatographic system is also equipped with a bioinert fraction collector, which enabled us to isolate specific components of interest (AAV8 monomers [F3], dimers [F2], and higher order aggregates [F1]) for further CD-MS analysis. Charge-Detection Mass Spectrometry (CD-MS) of AAV8. CD-MS is uniquely positioned to interrogate heterogenous macromolecules where charge state distribution and isotopic resolution cannot be achieved using conventional native MS. Furthermore, this single molecule technique has been successfully used to study higherorder aggregates . Our nSEC step used a mobile phase that is compatible with CD-MS. Therefore, the fractions were ready for direct nano ESI infusion. The nSEC fractions (Figure ) were infused using CE, ionized via nano ESI, and charge-detected. This workflow allowed us to measure individual ions and obtain molecular mass of empty, and particles with different amounts of genomic material. CE with a sheathless MS interface has shown proficiency for infusion of intact proteins . However, it is often difficult to maintain a stable spray using this interface due to its poor electrical contact at the capillary tip . In this workflow, CE with a sheath liquid MS interface was used as an infusion platform, as it delivers optimal spray stability with a fully controlled and stable flow-rate. nSEC fractions were infused at a pressure of 100 mbar, which corresponds to ~100 nL/min. Importantly, a controlled nano-flow rate can maximize the ionization efficiency in nano ESI by focusing the droplet emission zone toward the MS inlet . Automated CE infusions were performed using a capillary with a large inner diameter, which enabled direct ionization of AAV8 capsids without clogging problems. Clogging is a common issue in native MS and CD-MS experiments. As the infusion flow rate was fully controlled by our CE platform, the MS signal was stable, ensuring the robustness and reproducibility of our CD-MS strategy. Due to the extended acquisition time required by this MS-based method, long spray stability is required in CD-MS experiments. Importantly, sample consumption in CE is negligible, allowing us to continually introduce samples into the mass spectrometer for extended durations. Importantly, a neutral-coated capillary was used for infusions to avoid retention of these large virus-like particles in the capillary wall . Sulfur hexafluoride (SF6) was selected as a collision gas in the mass spectrometer. Heavy and polyatomic gases such as SF6 have shown proficiency at improving collisional cooling, focusing, and transmission of high m/z ions . rAAV preparations include AAV aggregates (dimers or other higher-order species), empty particles, partially-filled particles, and truncated particles. In addition, if the ssDNA genome is smaller than 4.5 kbs, rAAV particles with more than one genome can be identified . Figure shows the CD-MS spectrum of the nSEC fraction 3, which corresponds to the AAV8 single particles. The peak at 3.6 MDa represents a truncated version of the empty capsid (Figure ). Since the purchased AAV8 was packaged with a genome of approximately 2.5 kbs, half of the full-sized AAV genome, some particles were packaged with two copies. The peak at 4.5 MDa is consistent with a capsid filled with one copy of single-stranded DNA (ssDNA, ~800 kDa [Figure ). The peak at 5.2 MDa is consistent with a truncated version of the capsid that is packaged with two copies of ssDNA (Figure ). Here, we use the term "over-filled" to define a capsid that is packaged with two copies of ssDNA. The percentages of empty, filled, and over-filled capsids are shown in Table . The observed masses of the empty and over-filled capsids were approximately 100 kDa lighter than their theoretical masses. A reasonable explanation for these mass shifts is truncation of VP1/VP2 proteins. This proteolytic event in AAV capsids has been related to decreased potency . Importantly, truncation of the ssDNA genome can also occur . The charge versus mass to charge ratio scatter plot shows that the empty, filled, and over-filled capsids have very similar charges, indicating that the genetic material is packed inside the capsid (Figure ). As control samples, AAV8 empty and filled capsids were individually analyzed by CD-MS without nSEC fractionations (Figures ). While the empty particles were found to carry a small fraction of ssDNA, the filled particle was found to carry empty particles and particles with two copies of the genome. Table illustrates the percentages of empty, filled or over-filled in each capsid. To obtain the molecular masses of the AAV8 dimers and higher-order aggregates, we combined nSEC fractions 1 and 2 and analyzed them by CD-MS. CD-MS could not detect these large molecular species, which may be attributed to their extremely low abundances. However, prior detection using our nSEC strategy verified that AAV oligomers and higher-order aggregates were indeed present in the rAAV preparations, which confirms the complementarity between SEC and CD-MS. It has been widely accepted that empty capsids add to the total viral load, but they do not contribute to clinical efficacy . Figure illustrates low molecular weight AAV fragments (~250 kDa) that might be related to truncation of ssDNA or VP proteins. Interestingly, a significant portion of the empty capsid (95%) and a small portion of the filled capsid (3%) were detected in the aggregated nSEC fractions (Figure ). Importantly, empty capsids are more likely to aggregate than filled capsids in physiological solutions 47 . This phenomenon might be due to structural changes at the surface of the capsid caused by the absence of ssDNA, which increases electrostatic, hydrophobic, and polar interactions. This is evidenced by studies that employ IEX to purify empty capsids from their filled counterparts effectively . Although CD-MS analysis of the whole sample mixture without nSEC fractionation revealed similar results to the same fractionated sample mixture (Table and Figure ), our findings clearly suggest that aggregates contribute to the total viral load. They also suggest that our nSEC strategy is performing its function without or with negligible losses of the target compounds. Importantly, the primary role of our nSEC approach was to purify the sample prior to CD-MS analysis. It was achieved by removing unwanted aggregates, low molecular weight AAV fragments, and other impurities (Figures ). It is widely accepted that these contaminants can significantly impact the clinical safety, quality, and efficacy of biotherapeutic products. Thus, our nSEC strategy ensured that subsequent CD-MS analysis provides more accurate, consistent, reliable, and reproducible data. Furthermore, nSEC ensured that AAV characterization by CD-MS was more representative of the empty-full capsids, leading to a more accurate evaluation of total viral load, purity, and packaging integrity. Critical quality attributes such as purity, potency, safety, and accurate AAV characterization are required by regulatory agencies (e.g., FDA) . Thus, SEC purification prior to CD-MS analysis is needed to ensure that manufacturing processes meet these quality attributes.