The Sterility Tests - Microbiology Network

1 2 THE Sterility TESTSS cott SuttonMicrobiology Network , , NYUSABACKGROUNDThe compendial Sterility Test is not a test for product Sterility . This isnot a new, nor a particularly insightful observation. It has beenfrequently presented as a flawed test for its stated purpose in theliterature over the past 80 years. The test first appeared in 1932 ( , 1932) and included the basic features of the modern test two media, prescribed dilution scheme (for Bacteriostasis/Fungistasis or method suitability) and a defined incubation time. Theoriginal test had the media incubated for five days and allowed tworetests (all three had to fail to fail the test).

2 However, the basicstructure of the test was test has generated controversy as to its role in productquality testing for decades. While this is understandable, ithighlights a significant problem in Quality Control (QC)pharmaceutical Microbiology . We customarily use the compendialtest as finished product QC release Tests , but this is neither itsdesign nor intent. Those chapters in United States Pharmacopeia(USP) numbered less than 1000 (for example, the Sterility Test is7 USP chapter <71>) are referee Tests in other words they are inplace solely to demonstrate conformance to qualities specified inthe product monograph as described in the current NationalFormulary (NF) (the other part of the book).

3 A rigid interpretationwould have it that if the product is not described by NFmonograph, the test does not directly apply. In fact, the preface tothe internationally harmonized Sterility Tests reads: The following procedures are applicable for determining whether aPharmacopeial article purporting to be sterile complies with therequirements set forth in the individual monograph with respect tothe test for Sterility . In a similar vein, sterile finished dosage forms have the followingrequirement in USP (from <1>Injections): Sterility Tests Preparations for injection meet the requirementsunderSterility Tests <71> This has a nice symmetry the test states that it is applicable formeeting the requirements set forth in the monograph, therequirement being that the material meets the requirements of thetest.

4 Note that neither USP citation requires the finished product toactually be sterile, only that it meet the requirements of the test , one would have to conclude from a logical perspective thatthe test is not flawed for its intended purpose, that purpose beingto show that the material tested meets the requirements of the did we come to think that this test was designed to show thesterility of the product?We need something to demonstrate product Sterility . 21 CFR211 states the requirement: Special testing requirements.(a) For each batch of drug product purporting to be sterile and/orpyrogen-free, there shall be appropriate laboratory testing todetermine conformance to such requirements.

5 The test proceduresshall be in writing and shall be followed. Rapid Sterility Testing8 The difficulty, of course, is that there really is no way, given currenttechnology, to demonstrate Sterility of a batch. This imposessignificant validation issues. A way to satisfy this requirement isprovided in: Laboratory records.(a) Laboratory records shall include complete data derived from alltests necessary to assure compliance with established specificationsand standards, including examinations and assays, as follows: ..(2) A statement of each method used in the testing of the sample. Thestatement shall indicate the location of data that establish that themethods used in the testing of the sample meet proper standards ofaccuracy and reliability as applied to the product tested.

6 (If themethod employed is in the current revision of the United StatesPharmacopeia, National Formulary, AOAC INTERNATIONAL, Bookof Methods,{1} or in other recognized standard references, or isdetailed in an approved new drug application and the referencedmethod is not modified, a statement indicating the method andreference will suffice). The suitability of all testing methods used shallbe verified under actual conditions of use. So if we can cite a validated test we do not need to develop oneourselves. Thus the internationally harmonized Sterility Test ispressed into service as a product quality test even though that isneither its design nor its compendial Sterility Test has significant limitations as aproduct quality test.

7 We will discuss these limitations in the Sterility TESTST here are two different GMPs describing Sterility in the UnitedStates. The first is 21 CFR 211 and the second is the Biologics 21 CFR 610. By common consensus, the 21 CFR 211 CGMP looks to thecompendial Sterility Tests , while 21 CFR 610 describes a separatetest in 21 CFR The Biologics test is similar in fundamentalaspects to the compendial Sterility Tests . There is a finite (and small)The Sterility Tests9sample size and two recovery media are used, each with specifiedincubation conditions. Both types of types (compendial and Biologics ) so share some common limitations (see below).

8 The compendial Sterility Tests describe two separate types oftests (see McGuire and Kupiec, 2007 for a recent review). In the first,solution from a specified number of containers (volume andnumber determined by batch size and unit fill volume) is filteredthrough a filter of nominal pore size m. Recovery of viablecells from the filter(s) is performed by submerging the filter in oneof two recovery media followed by incubation at specifiedtemperatures for 14 days. The second test is a direct immersion ofthe product or suspensions into a suitable volume of the two mediato allow growth. The media are designed to support growth inaerobic, or growth in an environment of limited oxygen test requires demonstration that the specific method used issuitable for that US FDA Center for Biologics Evaluation and Research(CBER) version of the Sterility Test (21 CFR ) has been a sourceof some confusion for years, as it is almost the same as (but slightlydifferent from) the compendial test.

9 After the years of effort put intointernational harmonization of the compendial Sterility Test it washoped that CBER would just adopt it (with its flaws). However, theproposed draft (CBER, 2011) does not make this outcome seempromising. In the background material the statement is made thatthe USP test is acceptable as a validated test, but no mention ofthis position is made in the official text. In addition, where thecurrent test describes the media to be used, microorganisms usefulfor controls, incubation temperatures and duration, and mostimportantly sample size, none of these are described in the proposeddraft. All specific test methods have been removed to encourage theuse of validated Tests .

10 One has to wonder what they are to bevalidated against if there is no official comparator. These changeswill be discussed below. In any event, there is nothing in theproposed version that will prevent the use of the compendial Sterility Testing10 Limitations to the Sterility TestsAs early as 1956 Bryce published an article describing the two criticallimitations of this test. He put forward that the test was limited inthat it can only recognize organisms able to grow under theconditions of the test, and that the sample size is so restricted that itprovides only a gross estimate of the state of Sterility of the productlot (Bryce, 1956).