In the mid-1960s, Frances Bowman of FDA identified an organism that would penetrate the 0.45-μm-rated membranes that typically
were used for sterilizing filtration at that time (8). The organism, currently known as B. diminuta, American Type Culture Collection 19146, had the ability to distinguish between 0.22- and 0.45-μm-rated membranes (it readily
penetrated the latter) and, at concentrations higher than about 1 × 107 cfu/cm2, it penetrated 0.22-μm-rated membranes, demonstrating that because penetration was concentration-dependent, mechanisms other
than sieve retention were active. Although not the smallest organism known (smaller organisms had been known for decades),
B. diminuta generally was considered small enough to represent whatever smaller organisms were likely to be present in pharmaceutical
preparations. ASTM standard F 838, "Standard Test Method for Determining Bacterial Retention of Membrane Filters Utilized
for Liquid Filtration," is based on the use of B. diminuta as the challenge organism. The standard was originally approved in 1983 and was designed to determine the bacterial retention
characteristics of membrane filters for liquid filtration.
Bioburden organisms as models
Since then, however, some 25 cases have been noted wherein the 0.2/0.22-μm-rated membranes qualified by withstanding the requisite
B. diminuta challenge did not yield sterile effluent (9, 10). Obviously, B. diminuta does not serve as a universal model for all organisms. This finding has led some to advocate that the choice of the model
organism whereby a filter will be qualified for use as a sterilizing filter should be an organism native to the drug preparation.
In this view, B. diminuta suffers from the demerit of not being part of the drug preparation's bioburden. Be that as it may, selecting a component
of the bioburden as the model organism is not enough. The test organism must be amenable to identification and cultivation.
Its life stages must allow amply for its management in the necessary test manipulations. Above all, its susceptibility to
size diminution by contact with a given drug preparation will have to be investigated, and the kinetics and direction of the
morphological changes, if any, will require elucidation.
Insufficiency of the model organism
Summary points
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As previously stated, it had been believed that a sterilizing filter could be defined by way of its pore-size designation
as identified by integrity testing. Developments in filtration practices showed this belief to be too simplistic. What had
once seemed simple now is recognized as being quite complex. It was discovered that the conclusions based on pore-size ratings
were subject to modification by the physicochemical specificity of the organism-suspending fluid, the individuality of the
organism type in its size-changing response to the fluid, the possible change in pore size induced by the fluid, and the adsorptive
qualities of the filter resulting from its particular polymeric composition, all influenced by the filtration conditions in
their numerous varieties, but especially by the transmembrane pressure (11, 12).
A filter may not sterilize the same preparation under different filtration conditions, especially under dissimilar differential
pressures (13). A given membrane may or may not retain a particular organism type suspended in a different drug vehicle (8).
The organism type need not remain constant in size, but may alter in response to its suspending fluid (14–16). The effect
of the vehicle upon the polymeric membrane may cause a change in its pore size (17).
The certainty of obtaining sterile effluent requires far more than the identification of a sterilizing filter by a pore-size
rating. The variety of influences governing the outcome of an intended sterilizing filtration necessitates a careful validation
of the process, including the filter. The very drug preparation of interest, the exact membrane type, the precise filtration
conditions, and the prefiltration bioburden, including specific organism type(s) and number, should be used in the necessary
validation.
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