The validation of alternative microbiological methods for pharmaceutical testing is a very confusing topic, the more so as people use phrases such as "alternate sterility test" or "alternate microbial limits test." What we have to agree to from the outset is that in fact we are not looking for "alternate" compendial tests. What most people actually mean is that we are looking for an alternative (and usually more rapid) means to determine whether a sample would meet the requirements of the compendial test. In most cases, the major components of the compendial test will be respected, it is only one or two particular aspects of the test that will be changed to increase the reliability or speed of the test.
The sterility test by membrane filtration is an appropriate example. To simplify this discussion, the compendial test is assumed to be the standard against which others are to be compared. But what does it mean to have an alternate sterility test? Should 50 samples be taken rather than 20? Increasing the sample size will result in a more sensitive test but also a fundamental change in the compendial test. However, this is not supportive of the goal of finding a real-time test that will provide a high degree of confidence that the sample will pass the compendial test. One could change the nominal porosity of the filter, arguing that a smaller pore will retain "small bacteria" and so presumptively result in a more sensitive test. Few people are arguing for this modification. Finally, one could change the method used to determine whether the filter has retained viable cells.
The compendial test answers this question by requiring the contents of those samples to be filtered through a 0.45-μM filter and the filter to be placed in two different growth media (to increase the range of microorganisms potentially recovered), followed by an incubation period of 14 days. The recovery conditions (e.g., specific media, temperature, and incubation period) provide the parameters that determine the ability to recognize the presence of viable cells. An alternate test method may determine the presence of viable cells by testing for the presence of DNA, or the presence of ATP, a specific antigen, or the presence of an integral cell membrane and esterase activity (i.e., a vital dye). What is important to remember is that the specific question that must be answered is, Can we devise a better way to demonstrate the presence of viable cells on a filter than by growth to turbidity under specified conditions?An overwhelming advantage to rephrasing the question in this manner is that it provides us with testable predictions. For example, if we were to come across an antigen that was of particular use, then a serological test would need to demonstrate only that it recognized live (whole) cells rather than fragments and that it was able to "see" as many cells on the filter as does the compendial test. Similarly, a test using ATP bioluminescence, a vital stain, or polymerase chain reaction would only need to demonstrate that it was as sensitive to low numbers of cells as was the compendial test (in this case turbidity from a low inoculum on a membrane filter). Of course, it would be necessary for all these tests to demonstrate that the method was only measuring true events and not false positives from poor design.
Using this approach, three questions relate to this discussion:
These are the questions we wish to answer by an alternative means. It will serve us all well to remember that we can realize the benefits of a rapid answer while minimizing regulatory risk by clearly focusing our validation plans on the specific question of interest.