Pore-size ratings and validation
Until rather recently it was believed that the sterilization of fluids could be achieved by their filtration through a "sterilizing"
membrane whose proper and pertinent identity was confirmed by its pore-size rating, which was itself determined by bubble-point
measurement. This belief arose from the membrane's successfully withstanding a microbiological challenge of 1 × 107 cfu of B. diminuta per square centimeter of filter surface. On this basis, the membrane, in accord with FDAs definition, was considered a "sterilizing
Contrary to common belief, B. diminuta was not selected to be the model organism because it was thought to be the smallest microbe—smaller organisms were known
for decades (30). Rather, it was chosen as likely to be the smallest organism that might commonly be encountered in nonsterile
pharmaceutical preparations. Therefore, its filtrative removal was considered as indicating with a high probability that the
membrane used was a sterilizing filter. Using this type filter, identified by its bubble point, was assumed, with this caveat,
to ensure a sterile effluent.
Over time, it became evident that positive conclusions based on pore-size ratings were subject to modification by the physicochemical
specificity of the organism-suspending fluid, by the individuality of the organism type in its size-changing response to the
fluid, in the possible change in pore size induced by the fluid, and by the adsorptive qualities of the filter resulting from
its particular polymeric composition. All of these factors are influenced by the filtration conditions in their numerous varieties,
but especially by the transmembrane pressure.
A filter may not sterilize the same preparation under different filtration conditions, especially under dissimilar differential
pressures (31). A given membrane may or may not retain a particular organism type suspended in a different drug vehicle (3,
31). The organism type need not remain constant in size but may alter in response to its suspending fluid (32–34). The effect
of the vehicle upon the polymeric membrane may cause a change in its pore sizes (24). What had once seemed simple is now recognized
as being quite complex. Pore-size ratings alone are not sufficient to ensure the validation of a sterilizing filtration.
Given the above, it is surprising that The European Medicines Agency (EMEA) has stipulated certain requirements for a bioburden
confronting the sterilizing filter. Its 1996 Notes for Guidance states, "In most situations NFT 10 cfu/100 mL will be acceptable depending on the volume to be filtered in relation to the
diameter of the filter. If this requirement is not met, it is necessary to use a prefiltration through a bacteria-retaining
filter to obtain a sufficiently low bioburden" (35). In recommending the use of a bioburden-reducing filter when the level
of 10 cfu/100 mL is exceeded, the EMEA guideline states, "The type of bacteria-retentive filter and its pore size should also
be described in the application. Pore sizes of 0.22 μm are acceptable without further justification" (emphasis added). It would seem that validation by pore size is still acceptable to the EMEA.
Conclusions cannot be made regarding the sterile filtration of microorganisms unless methods of quantifying them by culturing
and counting are available. Organisms such as the L-forms, nanobacteria, and "viable but nonculturable" entities may not be
amenable to such analyses. Concerns about their presence may be justified but without the means to cultivate and count them,
it is impossible to attest to their complete absence. It follows that a sterilizing filter can be judged only by its performance
in the removal of identifiable and culturable organisms known to be present in the drug preparation (36).
Bubble points and "the largest pores"
An alternative approach focuses on measuring the set of "largest pores" of a filter. These are the least resistant to fluid
flow. They, if any, would be the most likely to permit organism passage. The measurement is of the pores' constricted diameters
rather than of their lengths. It is at these choke points that the size of a particle just large enough to be retained is
defined. The pore diameter varies along the length of the pore passageway and differs among the various pores of the filter;
hence, pore-size distributions characterize the microporous membranes.