Membrane filtration sterility assay: suitability test for amikacin eye drops 50 mg/mL

F. Saïag1, V. Lebreton1,2, A. Bourges1, M. Petit1, S. Vrignaud1
1 Pharmacy, University Hospital of Angers, France
2 MINT laboratory, INSERM U1066-CNRS 6021, Angers, France

Introduction
Amikacin eye drops are indicated for treatment of severe bacterial keratitis. These hospital preparations comply with good preparation practices. Physicochemical (drug concentration assay) and microbiological (sterility assay) controls must therefore be carried out.

The aim of this work is to develop the suitability test to validate sterility assay of amikacin eye drops.

Material and method
Membrane filtration method was performed according to European Pharmacopoeia (EP) 10th edition, monograph 2.6.1, using SteritestTM Symbio LFH pump (Merck-Millipore) and 0.45 µm cellulose ester membrane canisters (Merck-Millipore). Strains recommended by the EP were studied: Clostridium sporogenes, Pseudomonas aeruginosa, Staphylococcus aureus, Aspergillus brasiliensis, Bacillus subtilis and Candida albicans. In order to obtain inocula of less than 100 CFU, BioBall® SingleShot calibrated to 30 CFU (BioMérieux) were used. To avoid inhibiting effect of amikacin, Neutralizing Diluent Pharmacopoeia (DNP) (Oxoid) was added to eye drops dosed at 50 mg/mL. Culture medias used were thioglycolate (TG) and soya casein hydrolysate (SCH) (Oxoid). All these products comply with the EP.

Briefly, the test was performed as follows, a pre-wetting of the membrane with DNP, filtration of 10 mL of diluted eye drops in 80 mL of DNP and then rinsing with 90 mL of DNP. Subsequently, the membrane was rinsed by filtering 300 mL of 0.9% NaCl starting with 50 mL of 0.9% NaCl containing the inoculum. After this step canisters were filled with culture media. A negative control was performed at each session. Tests were performed in triplicate.

Note that C. sporogenes, P. aeruginosa and S. aureus strains were incubated for 5 days in TG, (30-35°C) while A. brasiliensis, B. subtilis and C. albicans strains were incubated for 5 days in SCH (20-25°C)

Results and discussion
At D3, growth of pathogens C. sporogenes, P. aeruginosa, S. aureus, A. brasiliensis, B. subtilis and C. albicans was visually detectable. After sending for identification to the bacteriology laboratory, analyses’results showed a monomorphic growth of each pathogen. Negative controls never visually showed microbiological contamination.

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Conclusion
Suitability test’s validation for amikacin 50 mg/mL eye drops was successfully completed. This allows to validate sterility assay for this preparation because it allows to conclude the absence of microorganism growth. This test can therefore be used routinely for microbiological control of these preparations.

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