Preliminary study to detect potential hydrogen peroxide permeability in medical packaging devices used for Advanced Therapy Medicinal Products

Objective
The rise of Advanced Therapy Medicinal Products (ATMPs) has forced healthcare facilities to adapt to handle their preparation. In this context, use of isolators remains currently limited due to the potential incompatibility of the primary packaging with hydrogen peroxide (H₂O₂) biocontamination. The objective of this study is to test a method for detecting H₂O₂ in order to identify potential permeability in CellSeal^® vials (made of cyclic olefin copolymer (COC)).

Method
Tests were conducted on cryopreserved vials filled with 4.6 mL of 1X PBS. The vials were thawed for thirty minutes at room temperature before being subject to the biodecontamination cycle. Three test series were performed (2 vials per test). Three samples were taken from each vial.
The method chosen for this detection is the Pierce™ Quantitative H₂O₂ Assay Kit from Thermo Scientific^®, which detects peroxides based on the oxidation of ferrous ions to ferric ions in the presence of xylenol orange. The oxidation forms a complex with the dye to produce a purple-colored product with a maximum absorbance at 560 nm. A Jenway 6850 spectrophotometer was used to measure absorbance. A calibration curve was prepared using Perox 35® solution (Sieve^®, Montalieu-Vercieu, France) for each test. The results are presented as mean ± standard deviation.

Results
The selected kit has a detection limit of 1.5 µM and a quantification limit of 5 µM. The dose range was obtained using Sieve’s Perox 35^® solution diluted to 1000 µM for each test. It includes 5 points: 5 µM, 15 µM, 30 µM, 50 µM, and 100 µM. The vials tested underwent an initial biocontamination cycle involving the spraying of 8 mL of 35% H₂O₂ with a contact time of 8 minutes, followed by a 4-minute rinse. In summary, two of the 6 vials tested were positive: one bottle with an H₂O₂ concentration outside the dose range and the second with an average concentration of 7.317 ± 0.006 µM, determined using a standard curve with an r² of 0.994.

Discussion
The vials tested underwent the standard thawing/reconstitution process with an isolator. The chosen method is effective and easily applicable. Given these initial results, it appears suitable for the detection of H₂O₂. The two positive results could be explained by a leak in the tubing; this issue will need to be addressed in future experiments. This preliminary work must be confirmed using a larger number of vials, but it could validate the use of the isolator for these devices and could be extended to other packaging types encountered with ATMPs.

Keywords: hydrogen peroxide, quantitative analysis, advanced therapy medicinal product

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