Impact of Time Out of Intended Storage and Freeze-thaw Rates on the Stability of Adeno-associated Virus 8 and 9

Abstract

There are an increasing number of clinical studies evaluating different adeno-associated virus (AAV) serotypes as vectors for gene therapy. Long-term frozen storage can maximize the stability of AAV. Freeze-thaw (F/T) cycles and exposures to room temperature (RT) and refrigerated conditions occur during manufacturing, labeling, and clinical use. In this work we exposed AAV8 and AAV9 at low and high concentrations to five F/T cycles compounded with RT and refrigerated holds in a 'daisy chain' time out of intended storage (TOIS) stability study, which may be a best practice in early development. We also evaluated the impact of 5 F/T cycles for multiple permutations of fast and slow cooling and rewarming rates. The quality attributes of AAV8 and AAV9 remained within acceptable ranges after the daisy chain TOIS and F/T rate studies. Potency and concentration were unchanged within method variability. There was a minor increase in non-encapsidated ('free') DNA released from AAV8 after F/T in a phosphate-buffered saline formulation. DNA release during F/T was minimized in a formulation with a low buffer concentration and was not detected in a formulation containing sucrose. We conclude that AAV8 and AAV9 have stability profiles that are suitable for manufacturing and clinical development.

Keywords: Adeno-Associated Virus (AAV); Degradation product(s); Deoxyribonucleic acid (DNA); Filling; Forced conditions; Gene Vector; Gene therapy; Protein formulation; Stability; Viral vector(s).