Resumo

Filtration is a common method of sterilizing drug product solutions. A sterilizing grade filter should be validated to reproducibly remove viable microorganism from the process stream, producing a sterile effluent. Brevundimonas diminuta (ATCC 19146) is a challenge microorganism for 0.2 µm rated filters. According to the definition of sterile filtration, a filter must remove at least 10⁷ cfu of B. diminuta per cm² of filter surface area. For the challenge bacteria testing, B. diminuta has to be properly grown and harvested before use. Additionally, identity, purity, cell size and viability of this challenge microorganism must be confirmed. The aim of this work was to evaluate the cultures of B. diminuta required to validate the sterilizing filtration of the radiopharmaceutical Fludeoxyglucose (18 F) (¹⁸FDG) produced at CDTN. B. diminuta was obtained from two different suppliers in lyophilized form. After reconstituting, stocks were maintained on tryptic soy agar (TSA). Several tests were performed in order to evaluate the challenge bacteria before use: direct inoculation, standard plate count, gram strain, sizing by microfiltration, microscopy, and biochemical identification. Viability of B. diminuta was confirmed by direct inoculation into the product and comparison with control. ¹⁸FDG showed to be nonbactericidal, since no more than a one log reduction in count was noted after 48 hours. The bacteria was checked for purity by the streak plate method on TSA plates at 30.0 ± 2.5°C and was examined for uniform colony morphology. Colonies were yellow-beige, slightly convex and shiny, indicative of B. diminuta. Gram strain results were identified as gram-negative rods. The size of the bacteria was evaluated by passing through a 0.45 µm rated membrane and total retention by the 0.2 µm rated filter, as required. Bubble point tests were performed to verify membrane integrity before and after use. Stock suspensions of the cultured microorganism were submitted to biochemical assays. The preparation and maintenance of B. diminuta from different suppliers provided suspensions equally suitable for the bacterial challenge testing. Appropriate procedures were developed to grow and to evaluate the strains of B. diminuta at the radiopharmacy unit of CDTN, supporting future work on sterilizing filtration process validation.