AAV process intensification using high-salt lysis and salt-tolerant endonuclease

The increasing demand for recombinant adeno-associated viruses (rAAVs) in high-dose clinical trials and therapeutic applications requires increased efficiency and scalability. High salt concentrations (>150 mM) in production enhance rAAV yields and potency but can negatively impact DNA digestion [1] [2]. This study evaluated the effect of four detergents (Polysorbate 20, Triton™ X-100, Deviron® C16, Deviron® 13-S9) at 150 mM and 500 mM NaCl concentrations on rAAV5 and rAAV2 vector yields and protein expression potency. Higher rAAV5 titers were observed with all detergents at 500 mM NaCl compared to 150 mM NaCl. Similarly, rAAV2 titer was 10-fold higher at 500 mM NaCl, than 150 mM. However, under these high salt conditions, standard endonuclease DNA digestion was ineffective. A protein-engineered, salt-tolerant endonuclease was developed to enable efficient DNA digestion in salt concentrations up to 1,000 mM NaCl. When combined with high-salt lysis, this endonuclease improved rAAV yields and titers while meeting requirements for DNA clearance. The use of high salt concentrations with Benzonase® Salt Tolerant endonuclease enhances the productivity of rAAV processes.

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