Quantification of adeno-associated virus aggregation using asymmetrical flow field-flow fractionation

Asymmetrical flow field-flow fractionation (AF4) is a separation technique using an unpacked separation channel instead of a packed column. Lack of a stationary phase provides unique advantages, which are: no shear forces, matrix interaction-free sample separation, fast separation times and great flexibility with respect to the choice of solvent. Also, AF4 offers a wide separation range from 1 kDa/ 1 nm up to several MDa/ 10 um. Thus, a complete picture of the aggregation phenomena is provided, including monomer, soluble and insoluble aggregates and even the subvisible particle fraction.

In this application note, Postnova presents data on separation of AAVs and their aggregates using AF4 and measurement of their radius of gyration (Rg or root mean square radius, R.M.S.) by multi angle light scattering (MALS). The combination of cross flow and channel flow causes size separation over the course of the analysis, with smaller particles eluting before larger particles, including aggregates. As the particles are eluted from the channel they flow sequentially through the connected detectors.