Understanding the effect of Benzonase endonuclease on downstream purification of adeno-associated viral vector (AAV) manufacturing

Adeno-associated viruses (AAVs) are an emerging modality as promising vectors for gene therapy. The manufacturing process involves cell expansion, infection and subsequent purification processes, which involves adventitious viral inactivation, cell lysis, followed by several chromatography and ultrafiltration/Diafiltration steps. This work examined the use of benzonase® endonuclease in the clarified lysate for nucleic acid (DNA) digestion and its impact on downstream processes that involve affinity purification and polishing chromatography.
The optimized conditions for benzonase® endonuclease were chosen to maximize the enzyme activity on DNA digestion and the effect of benzonase® treatment on the performance of the affinity step was studied in combination with several wash and elution conditions. The implications of using benzonase® treatment on the lifecycle of the affinity resin was also evaluated and the implications of this treatment step on impurities at the drug substance level was assessed. A guideline for an optimized process that balances AAV recovery, AAV purity, processing time and manufacturability is provided based on the outcomes of this work.