Single-use chromatographic clarification to eliminate endonuclease treatment in production of recombinant adeno-associated viral vectors

Abstract

In current manufacturing processes for recombinant adeno-associated viral vectors (rAAV), endonuclease treatment is used prior to depth filtration to break down host cell DNA that is released from cells post-lysis. Without this treatment, traditional depth filters exhibit negligible clearance of host cell DNA, a key process-related impurity. In this work, we evaluate single-use chromatographic clarification using Harvest RCTM filters (3 M) to process rAAV8 and rAAV9 HEK293 cell culture lysate with and without endonuclease pretreatment. We demonstrate endonuclease-free clarification for both serotypes with yield >90 % and up to 3 log reduction of host cell DNA, from >104 ng/mL to <50 ng/mL, matching or exceeding the filtrate quality in traditional depth filtration with 100 U/mL of endonuclease added during lysis. The addition of 100 mM of salt in the harvest buffer is also shown to improve filtration throughput, prevent filter clogging, and limit rAVV binding to the filter device. Finally, a mechanistic cake-fiber fouling model is shown to be a good representation of the Harvest RCTM filtration mechanism. The endonuclease-free clarification approach enables cost savings of USD 100,000 per 500 L batch, equivalent to the cost of cGMP-grade endonuclease at this scale. The benefits of endonuclease-free clarification further include easier raw material sourcing as well as eliminating the need for a residual endonuclease assay in rAAV manufacturing processes.