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

Background
In our pharmacotechnics unit, the sterility test of 5% vancomycin eye drops is carried out by direct seeding on Trypticase-Soy and Sabouraud agars. However, the European Pharmacopoeia (EP) recommends the membrane filtration method whenever the nature of the product allows it.

Objective
Develop and validate the sterility test method by membrane filtration of 5% vancomycin eye drops.

Material & Methods
The tests are carried out according to EP 10th Ed. “2.6.1. Sterility” using a Steritest^® Symbio LFH peristaltic pump (Merck-Millipore) with a fertility test (positive control) and a test for sterility each comprising 3 steps : pre-wetting of the membrane, filtration of eye drops (8 mL), rinsing of the membrane. The membranes are then incubated in culture media. The canisters used have a PVDF membrane (porosity < 0.45µm). Aliquots of 6 microbial strains are prepared from BioBall^® Multi-shot 550 beads (Biomérieux) and introduced during the rinsing step. The microbial growth in the canisters must testify to the effectiveness of the rinsing. Initially, the protocol used is a transposition of that validated for ceftazidime 5% eye drops in our unit. After discussion with different centers, other tests are carried out by modifying the following parameters: dilution of the eye drops, pump speed, composition of the rinsing fluid.

Results
Transposition alone of the validated protocol for ceftazidime 5% eye drops does not allow validation of the method with a non-reproducible growth of Staphylococcus aureus. The addition of an eye drop dilution step allows reproducible growth of S. aureus but no growth of Bacillus subtilis and Clostridium sporogenes. The tests carried out after modifying the pump speeds do not show any growth of B. subtilis and C. sporogenes. Trying a different rinsing fluid does not allow growth of B. subtilis and non-reproducible growth of C. sporogenes. The growths are reproducible for the other 4 microbial strains tested.

Discussion & Conclusion
The suitability test carried out according to EP validates the method for 4 of the 6 microbial strains tested. In view of our results, comparable to those of other centers, B. subtilis seems to be the critical strain for sterility testing of vancomycin 5% eye drops. The dilution step is an interesting step but the use of NaCl should be preferred to water for injection so as not to lyse the microbial strains. The use of a rinsing fluid incorporating a neutralizer does not allow complete inhibition of vancomycin. An adsorption of vancomycin on the membrane would be the main hypothesis of the absence of growth for B. subtilis and C. sporogenes.

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