Method validation in preservation of sterile agar plates beyond the manufacturers’ recommendations for use in a compounding unit

Context
The use of agar plates (AP) was considered for microbiological monitoring of our class A isolator, in accordance with current good compounding practices. Per production campaign, we only use 2 out of the 10 agar plates packed in a triple sterile packaging. Our supplier does not guarantee their fertility beyond 2 days in these conditions; the use of a gastight box could extend their conservation over 5 days.

Objective
Study the fertility of the AP beyond the 2 days recommended by the supplier thanks to a hermetic device initially intended for the microbiological culture in anaerobic conditions.

Material and methods
The AP were placed inside our isolator at room temperature (22°C ± 2°C) in a gastight box (BD^® Gazpak EZ) (n=15) and outside the isolator (n=15) for 5 days after opening the primary packaging, then exposed for 4 hours in ambient air before inoculation.
Fertility test was performed with 5 micro-organisms according to the European Pharmacopoeia and manufacturer’s quality test: 3 bacteria (Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus) and 2 fungi (Aspergillus brasiliensis, Candida albicans). Low inocula (<100 CFU) were plated in triplicate. Control agar plates were used to determine the agar coverage rate (ratio of the number of test AP CFU / number of control AP CFU). Change in AP weight was measured as a secondary endpoint to assess water loss in the agar plates. The observed variations were compared using a Student’s t test ( ≤ 0.05)

Results
The mean recovery rate of each germ is higher than 70% with P.aeruginosa = 158%, B.subtilis = 94%, S.aureus = 100%, A.brasiliensis = 83%, and C.albicans = 79%. Outside the isolator, a significant loss of the average weight of the AP was observed 5 days after opening the primary packaging (p = 0.02), contrary to those kept in the hermetic box (p = 0.43). This weight loss is also significant outside the isolator after exposure for 4 hours in ambient air (p < 0.001), in contrast to the AP exposed inside the isolator (p = 0.43).

Conclusion and relevance
The results of the fertility test are congruent with the expected results suggesting that the use of an airtight plate allows preserving the fertility of agar plates 5 days after opening the primary packaging in our study conditions.

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