P-BT-1 - Qualification of a Residual Peracetic Acid Assay for Amnion Patches Undergoing Supercritical CO2 Sterilization

Human amnion patches, derived from placental membranes, are increasingly used in wound healing, ophthalmology, and regenerative medicine due to their regenerative properties. However, conventional sterilization methods can compromise their biological function. Supercritical carbon dioxide (scCO₂) sterilization has emerged as a gentler alternative that maintains tissue structure and bioactivity. The sterilization process uses the disinfectant additive NovaKill, a peracetic acid (PAA) based additive, in conjunction with scCO₂ to achieve microbial inactivation. To ensure residual PAA levels remain within safe limits post-sterilization, the NovaPAK™ kit from NovaSterilis was qualified to accurately quantify PAA in amnion patches. This qualification study evaluated the assay’s performance based on precision, accuracy, linearity, and specificity. Additionally, the extraction process for the patches was optimized to improve measurement reliability and ensure consistent, safe application in clinical settings.

Study

Design/Methods:

The NovaPAK™ kit was used following manufacturer protocols. Intra-assay precision was assessed with six replicates of low (2 ppm) and high (5 ppm) PAA concentrations in a single run. Inter-assay precision involved testing the same concentrations over five days by two operators using two microplate readers, totaling 15 replicates per level. Accuracy was assessed at 10 ppm, 5 ppm, and 2.5 ppm across two runs by two operators. Linearity was evaluated using an 8-point serial dilution (10 to 0.585 ppm) in triplicate. Specificity was tested using external PAA and a non-PAA acid. The extraction process was optimized by varying patch size, extraction volume, temperature, and time to improve PAA recovery after scCO₂ sterilization.

Results/Findings:

All results met acceptance criteria. Inter- and intra-assay precision showed %CV ≤ 20, and accuracy tests yielded 50–150% recovery. Linearity tests had %CV ≤ 20 across concentrations, with an R² ≥ 0.95. Specificity was confirmed by detecting external PAA and not HCl. The optimal extraction process was 72 hours at 37°C with a volume-to-surface area ratio of 6 cm²/mL, closely mimicking physiological conditions for reliable PAA measurement.

Conclusions:

The comprehensive qualification data generated demonstrated that the residual PAA assay is both reliable and accurate, providing confidence that the results generated are consistent, reproducible, and reflective of the true PAA concentrations in the tested samples. This residual peracetic acid assay can be a method used to quantify PAA in other biological samples that undergo the scCO₂ sterilization process, providing safe products to consumers.