Mechanistic Studies and Formulation Mitigations of Adeno-associated Virus Capsid Rupture During Freezing and Thawing: Mechanisms of Freeze/Thaw Induced AAV Rupture.

Gene therapies delivered using adeno-associated virus (AAV) vectors are showing promise for many diseases. Frozen AAV drug products are exposed to freeze-thaw (F/T) cycles during manufacturing, storage, and distribution. In this work we studied the mechanisms of AAV capsid rupture during F/T. We found that exposure to interfaces, exacerbated by F/T, and the mechanical force of excipient devitrification correlated with AAV capsid rupture during F/T. There was no impact of pH shifts, cryo-concentration, or cold-denaturation. Results were similar for AAV8 and AAV9. With these mechanistic insights we identified three formulation mitigation approaches. Addition of ≥0.0005% w/v poloxamer 188 (P188) eliminated substantial recovery losses (up to ∼60% without P188) and minimized rupture to ≤1% per F/T cycle. Elimination of exothermic devitrification events during rewarming, either by formulating with a low buffer concentration, or by adding a cryoprotectant further reduced rupture during F/T. Rupture of AAV9 was <0.2% per F/T cycle in a formulation with 1 mM phosphate, 4.4 mM dextrose, electrolytes, and 0.001% P188 at pH 7.2. Rupture of AAV8 was not detected when formulated with 4% sucrose, 100 mM salt, and 0.001% P188 at pH 7.4. These results provide insights into effective strategies for stabilizing AAVs against rupture during F/T.

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

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.

Development of a stable lyophilized adeno-associated virus gene therapy formulation.

Adeno-associated viruses (AAV) are among the most actively investigated vectors for gene therapy. Supply of early clinical studies with frozen drug product (DP) can accelerate timelines and minimize degradation risks. In the long-term, logistical challenges of frozen DP may limit patient access. In this work, we developed a lyophilized (freeze-dried) formulation of AAV. The mass concentration of AAV is typically low, and AAV also requires a minimum ionic strength to inhibit aggregation. These factors result in a low collapse temperature, which is limiting to lyophilization. Mannitol crystallization was found to cause extensive degradation and potency loss of AAV during the freezing step. With further development, we determined that AAV could be lyophilized in a sucrose and citrate formulation with a more desirable high glass transition temperature of the dried cake. An optimal residual moisture range (1-3%) was found to be critical to maintaining AAV8 stability. Glycerol was found to protect AAV8 from over-drying by preventing capsid damage and genome DNA release. A lyophilized formulation was identified that maintained potency for 24 months at 2-8 °C, indicating the feasibility of a dried formulation for AAV gene therapy.

Quantitation of Trace Levels of DNA Released from Disrupted Adeno-Associated Virus Gene Therapy Vectors.

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.

Revising how the computer program CERVUS accommodates genotyping error increases success in paternity assignment.

Genotypes are frequently used to identify parentage. Such analysis is notoriously vulnerable to genotyping error, and there is ongoing debate regarding how to solve this problem. Many scientists have used the computer program CERVUS to estimate parentage, and have taken advantage of its option to allow for genotyping error. In this study, we show that the likelihood equations used by versions 1.0 and 2.0 of CERVUS to accommodate genotyping error miscalculate the probability of observing an erroneous genotype. Computer simulation and reanalysis of paternity in Rum red deer show that correcting this error increases success in paternity assignment, and that there is a clear benefit to accommodating genotyping errors when errors are present. A new version of CERVUS (3.0) implementing the corrected likelihood equations is available at http://www.fieldgenetics.com.