Native Size Exclusion Chromatography (nSEC) Conditions. Ammonium acetate solution 7.5M (A2706) was purchased from Sigma-Aldrich. AAV8 empty (AAV8-Empty [2E+13 vg/ml]) and full (AAV8-CMV-GFP [2E+13 vg/ml]) capsids were purchased from VIROVEK. The theoretical mass of the empty capsid was expected to be ~3.7 MDa while the theoretical mass of the filled capsid was expected to contain one copy of single-stranded DNA (ssDNA, ~2.4 kb or 800 kDa). AAV8 empty and full capsids were mixed (1:1, v/v). The AAV8 mixture was injected into an1260 Infinity II Bio-inert LC system (Agilent Technologies). The mobile phase was 200 mM ammonium acetate. The AAV8 mixture was fractionated on an AdvanceBio SEC column (300 x 7.8 mm; 2.7 µm; 500 Å [Agilent Technologies]) using isocratic elution at a flow rate of 1.0 mL/min. Fractions were collected from ~4-10 min at 1 min intervals. A volume of 80uL was injected into the chromatographic system. The elution profile was monitored by UV absorbances at 280 and 260 nm and the Agilent 1260 MALS at 20 different angles. The WinGPC software (Agilent Technologies) was used to process the data. The nSEC fractions were collected using a 1260 Bio FC-AS fraction collector (Agilent Technologies). The temperature of the fraction collector was set to 4°C. Each nSEC fraction was then concentrated/desalted using a 100kDa molecular weight cutoff filter for subsequent CD-MS analysis. The concentrated/desalting procedure was performed 1 time (12,000 × g for 5 min at 4°C) to minimize salt effects. UV absorbance measurements at 260 and 280 nm were used to determine the ratios of empty and full capsids (monomeric and dimeric assemblies). The percentages of dimeric assemblies in the AAV sample mixture were calculated using the formulae below. ). A calibration curve in "high mass" was used to assign charge states. Carbonic anhydrase, alcohol dehydrogenase, pyruvate kinase, and β-galactosidase were used to construct the calibration curve. Processing templates were used to interpretate the data and calculate the AAV percentage. After processing the data, the resolution was adjusted to 0.001.