Development and validation of sterility test method by membrane filtration of ceftazidime 5% eye drops

Introduction
The sterility test of ceftazidime 5% eye drops prepared in our unit is currently performed by direct inoculation on Trypticase soy agar and Sabouraud agar media. According to the European Pharmacopoeia (EP), the membrane filtration method is chosen when the nature of the product permits.
Objective : To develop and validate the sterility test by membrane filtration of ceftazidime 5% eye drops.

Material & Method
The tests are performed according to the EP 10th Ed. "2.6.1. Sterility" using a Steritest^® Symbio LFH vacuum pump (Merck-Millipore). The steps are pre-wetting of the membrane, filtration of the eye drops solution, rinsing of the membrane, addition of the culture medium and incubation of the membrane. Bibliographical research and an exchange with different centers allow to determine three tests to be carried out on three eye drops each, using a strain of Pseudomonas aeruginosa, considering the ceftazidime spectrum of activity , and to be compared to a positive control. The optimal parameters thus determined are validated with six strains as defined in the EP (see method applicability test) and by three operators. Aliquots of microbial strains are prepared from BioBall^® Multi-shot 550 beads (Biomérieux) and diluted in the rinsing fluid at the rinsing step.

Results
The volumes of rinsing fluid and pump speeds are set according to the supplier’s recommendations. The two main parameters are the type of rinsing fluid and membrane. Two rinsing fluids were tested: peptone water (F1) or peptone water + polysorbate 80 (F2); and two membranes: cellulose ester (CE) or polyvinylidene fluoride (PVDF). For each test, 10 ml of eye drops solution are filtered. The 3 tests performed are (1) F1 fluid + EC membrane; (2) F1 fluid + PVDF membrane and (3) F2 fluid + PVDF membrane. A growth of Pseudomonas aeruginosa comparable to that of the positive control is observed at D5 on one test (1) and on the 3 tests (2) and (3). Method (3) is chosen for the applicability test. A microbial growth comparable to the positive controls is observed at D5 for each of the 6 microbial strains and for each operator.

Discussion & Conclusion
Test (3) is preferred because the polysorbate 80, surfactant, favors the elimination of antimicrobial activity and the thinner PVDF membrane (120µm) than the EC one (150µm) reduces the risk of ceftazidime adsorption. This risk is also reduced thanks to the pre-wetting which saturates the pores of the membrane and to the rapid filtration of the eye drops. The method is validated and will be used routinely to control the sterility of ceftazidime 5% eye drops. The next steps are the realization of a microbiological stability study of ceftazidime 5% eye drops and the development of the method on other antibiotic eye drops.

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