Adeno-associated virus (AAV) vectors for gene therapy have potential to provide a durable treatment response for a number of diseases with unmet need. DNA is released from AAV capsids at high temperatures. Less is known about DNA release that may occur under conditions relevant to clinical and commercial manufacturing, storage, and distribution. In this work we developed and applied a sensitive fluorescent dye-based method to quantitate trace levels of DNA released from AAV capsids. The method was used to characterize the impact of manufacturing process steps on the increase (up to 1.5%) and removal (down to 0.2%) of free DNA. Free DNA increased by 0.3% per day at 37 °C and by 0.4% per freeze/thaw cycle in a phosphate-buffered saline formulation. When stored for 2 years at different temperatures, free DNA remained low (<0.6%) at both ≤ -60 °C and at 2-8 °C but was higher (2.6%) when the same sample was stored at -20 °C. The dye-based method may be used to further characterize release of free DNA for different processes, formulations, and stress conditions. Overall, release of free DNA was a relatively minor degradation pathway under the conditions studied in this work.
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