Professionals in bacterial retention validation are well aware that regulatory agencies and the industry universally recognize Brevundimonas diminuta (formerly Pseudomonas diminuta) ATCC® 19146™ as the standard challenge microorganism. This bacterium is exceptionally small, once considered the smallest known bacterium, with a diameter of approximately 0.3-0.4 μm and a length of 0.6-1.0 μm. As a water-borne, Gram-negative organism, it is broadly representative of potential contaminants encountered during pharmaceutical manufacturing. An additional rationale for its selection is that it is non-pathogenic and can consistently produce reproducible, single-cell populations with stable size and morphology under standard culture conditions.
The size of the challenge microorganism must be confirmed by its ability to penetrate a 0.45 μm pore-size membrane, which serves as the positive control for each challenge test. Three separate lots of 0.22 μm (or 0.2 μm) test filters and a 0.45 μm control filter must be challenged in parallel within a single, integrated test system. Under standard culture conditions, B. diminuta at high challenge levels (typically ≥10⁷ CFU) should show limited breakthrough through the 0.45 μm membrane.
However, there are circumstances in which B. diminuta may not represent the worst-case scenario. For instance, if microorganisms smaller than B. diminuta are detected in the actual drug product during manufacturing, consideration should be given to using such organisms for the bacterial challenge test. If an alternative microorganism is employed, comprehensive documentation must be provided to justify its use. The alternative organism must be sufficiently small to challenge the retention capability of sterilizing-grade filters and must simulate the smallest microorganism found in the product. Accordingly, an alternative challenge microorganism may be used in validation studies, provided that the pre-filtration bioburden level is determined to be more relevant. Whenever feasible, the indigenous bioburden should be characterized, enumerated, and identified, as these organisms may possess the potential to penetrate sterilizing-grade filters. The morphology of the isolated microorganisms must also be taken into account.
Therefore, it is recommended that manufacturers conduct routine microbial monitoring and analysis to assess whether microorganisms smaller than B. diminuta are present in the environment or during production, thereby providing data-driven support and justification for the selection of the challenge microorganism.