More and more transformative gene therapies (GTx) are reaching commercialization stage and many of them use Adeno Associated Viruses (AAVs) as their vector. Being larger than therapeutic antibodies, their size variant analysis poses an analytical challenge that must be addressed to speed up the development processes. Size exclusion chromatography (SEC) can provide critical information on the quality and purity of the product, but its full potential is not yet utilized by currently applied columns that are (i) packed with relatively large particles, (ii) prepared exclusively in large formats and (iii) built using metal hardware that is prone to secondary interactions. In this paper, we investigate the use of state-of-the-art sub-3 µm particles to address existing limitations. A prototype 2.5 µm column was found to deliver superior kinetic efficiency, significant reduction in run times and increased resolution of separations. No evidence for shear or sample sieving effects were found during comparisons with conventional 5 µm columns. Moreover, use of low adsorption hardware enabled the application of a wide range of mobile phase conditions and a chance to apply a more robust platform method for several AAV serotypes. The resulting method was tested for its reproducibility as well as utility for critical quality attribute assays, including multiangle light scattering based (MALS) measurements of size and molar mass. Thus, a new tool for higher resolution, higher throughput size variant analysis of AAVs has been described.
Keywords: AAVs; Adeno associated virus; Gene therapy; SEC; Size exclusion chromatography; Size variants analysis.
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