Targeted Optimization of AAV8 Upstream Process for Enhanced Downstream Performance

Several recombinant AAV (rAAV)-mediated therapies have been approved so far, and many more are in clinical trials, yet developing an efficient rAAV process platform remains a challenge. First obstacle arises from frequent practice of upstream (USP) and downstream (DSP) process being developed separately. USP optimization activities are usually focused on achieving high viral titer, which does not always translate into maximal purity and recovery in DSP. Second significant challenge is to bridge USP and DSP into a single bioprocess, due to lack of appropriate analytical methodology capable of producing reliable and unbiased results on both sides of the bridge. Furthermore, rAAV-based gene therapy vectors require the removal of process-specific combined with product-specific impurities, as they represent serious safety threats. Here we showcase the example of AAV8 USP optimization directed towards good performance in DSP. AAV8 is produced in HEK suspension cells via plasmid transfection. Based on the screening results, the best performing set of conditions was successfully scaled up to a 5L stirred-tank bioreactor. Harvested material was then evaluated in DSP, comprised of pre-capture, and two ion-exchange chromatographic steps on CIM monolithic columns. As bridging analytics of choice, we relied on PATfix platform, (d)dPCR and Comassie Bradford protein assay measurements for thorough assessment of relevant parameters such as titer, percentage of full AAV8 capsids and total protein concentration.