2.2. Size-Exclusion Chromatography Multiangle Light Scattering (SEC–MALS)

Chromatographic separation of the rAAV samples was achieved using a Superose 6 increase (10/300) with an isocratic flow rate of 0.8 mL/min and phosphate-buffered saline as the mobile phase. An OMNISEC system (Malvern Panalytical Ltd., Malvern, UK) consisting of an OMNISEC RESOLVE (pump, autosampler, and column oven) and an OMNISEC REVEAL (refractive index, UV/Vis-PDA and right-angle/low-angle light scattering detector) was used to acquire the sample chromatograms. The samples were maintained at 4 °C in the autosampler prior to injection. The column oven and detector module were maintained at a constant 30 °C during this work.

Following separation, the samples were processed using a compositional analysis designed to determine the concentration and molecular weight of two distinct components within a sample. For the compositional analysis to work, it is necessary to know the refractive index increment (dn/dc) and extinction coefficient (dA/dc) of both components. In this case, the dn/dc of the capsid and the ssDNA is well known. The dA/dc for the capsid can be measured using OMNISEC, and the dA/dc for the ssDNA is calculated from the sequence.

Total particle (Equation (1)), full particle (Equation (2)), and empty particle (Equation (3)) concentration can all be obtained using the following equations:

where Conc_Capsid is the concentration of the capsid in mg/mL as calculated, N_A is Avogadro’s number, Mw_capsid is the molecular weight (g/mol) of the capsid as calculated, Conc_DNA is the concentration of the DNA in mg/mL as calculated, and Mw_{Seq DNA} is the molecular weight of the ssDNA from the sequence. Therefore, using these calculated particle concentrations, the percentage of full rAAV5 in a sample can be easily derived. The compositional analysis of SEC–MALS data in this study is based on a simplified model where capsids are treated as empty or filled only.