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Sterility testing

Sterility testing is a microbiological test that examines samples of a material for viable contaminating microorganisms. ICH Q4B Annex 8(R1), which FDA publishes as guidance, evaluated three pharmacopoeial sterility texts, Ph. Eur. 2.6.1, JP 4.06 and USP <71> Sterility Tests, and recommends them as interchangeable in the ICH regions under the conditions it lists.[1],[2],[3]

What does a compendial sterility test look for?

Parveen and colleagues, writing from FDA's Center for Biologics Evaluation and Research in 2011, described the sterility testing method then set out in 21 CFR 610.12 and in USP <71> as based on the observation of turbidity in liquid culture media due to growth of potential contaminants.[4]

The compendial media in their comparison were fluid thioglycollate medium and tryptic soy broth, used in both the membrane filtration and the direct inoculation forms of the compendial method.[4]

FDA's 2004 guidance on aseptic processing names USP <71> Sterility Tests as the principal source used for sterility testing methods, including information on test procedures and media.[5],[3]

How do membrane filtration and direct inoculation compare?

In the evaluation by Parveen and colleagues, the compendial membrane filtration method was significantly more sensitive than two automated carbon dioxide monitoring culture systems, which the authors considered possibly applicable as alternatives to the compendial direct inoculation method for products without preservatives or antimicrobial agents.[4]

Bathgate and colleagues analysed ten years of sterility test results and found no overall difference between membrane filtration and direct inoculation in the time required for visible growth of contaminants. Growth appeared earlier in products without preservative or antimicrobial substances, and regardless of product or method, limiting incubation to seven days would have missed an unacceptable proportion of contaminants.[6]

The 2004 FDA guidance states that where growth is inhibited during method validation, modifications such as increased dilution, additional membrane filter washes or inactivating agents should be used, and that validation should show the method does not provide an opportunity for false negatives. ICH Q4B Annex 8 adds that diluting and rinsing fluids should not have antibacterial or antifungal properties.[5],[2]

What are the statistical limits of testing a sample?

FDA's aseptic processing guidance states that sterility tests are limited in their ability to detect contamination because of the small sample size typically used. Citing USP, it states that the sampling plan only enables the detection of contamination in a lot in which 10% of the units are contaminated about nine times out of ten.[5]

The guidance gives an illustration: a 10,000-unit lot with a 0.1 percent contamination level, tested using 20 units, has a 98 percent chance of passing.[5]

Because of that limited sensitivity, the guidance regards any positive result as a serious current good manufacturing practice issue to be thoroughly investigated, and it states that samples should represent the whole batch, including the beginning, middle and end of the aseptic processing operation.[5]

For one category of liquid products in batches of more than 500 containers, ICH Q4B Annex 8 considers the texts interchangeable if the minimum number of containers selected is 20 or 2 percent of the total, whichever is lower.[2]

What does the U.S. regulation for biological products require of the method?

21 CFR 610.12, as amended in 2012, requires that a sterility test for a biological product be appropriate to the material so that the material does not interfere with the test, and that it be validated to show it can reliably and consistently detect viable contaminating microorganisms.[1]

The regulation distinguishes culture-based methods, whose written procedures specify media composition, growth-promotion testing and incubation time and temperature, from non-culture-based methods, whose procedures specify test components, parameters with acceptance criteria and controls. For non-culture-based methods it requires appropriate controls within the test itself.[1]

If an initial test indicates microorganisms, the regulation states that the product does not comply unless a quality control unit investigation definitively ascribes the result to laboratory error or faulty materials, in which case the test may be repeated once by the same method.[1]

What are rapid microbiological methods?

The 2004 FDA guidance states that other suitable microbiological methods, such as rapid test methods, can be considered for finished product release testing after it is demonstrated that they are equivalent to or better than traditional methods.[5]

Parveen and colleagues found an ATP bioluminescence system detected all test microorganisms faster than the compendial method and was acceptable as an alternate method taking 5 days, compared with the 14 days of the compendial method. England and colleagues, testing 118 challenge organisms, reported that the compendial USP <71> method detected significantly more than one automated blood culture system, which they judged suboptimal for product sterility testing.[4],[7]

Wallace and colleagues describe a method that uses an automated culture system as an enrichment step followed by PCR detection of viable microorganisms, reporting a time to detection of 3 days and a limit of detection of 1 colony forming unit.[8]

IJzerman-Boon and colleagues note that USP presents two approaches for showing that an alternate qualitative microbiological method is non-inferior to a compendial method, and report simulations indicating that the rate-based approach is not suitable while the approach based on most probable number estimates is appropriate.[9]

How does this relate to a PepGenex certificate?

The sterility-related test line on a PepGenex certificate of analysis is Rapid Sterility Screening (PCR), reported as Not Detected. This screening is a detection result for the sample tested and does not by itself establish the sterility of every unit or a validated sterility assurance.

Limitations

A negative result applies to the units tested. Because FDA describes sterility tests as limited by their small sample size, a passing result does not show that every unit in a lot is free of viable microorganisms.[5]

Sterility and endotoxin appear as separate entries among the pharmacopoeial tests in ICH Q6B, and a sterility result carries no information about endotoxin.[10]

The comparative studies cited here evaluated specific commercial systems with specific organisms and matrices; their results are not general statements about every rapid method. The Wallace study was online ahead of print when read.[4],[7],[8]

This page describes what the cited documents say. It is not a statement that any PepGenex material has been manufactured, tested or released under any of them.

Compound profiles

Research peptide profiles in this library. What this page describes applies to peptide lots in general; none of these profiles reports a result of it for any lot.

References

  1. 21 CFR 610.12, Sterility (current edition; 77 FR 26174, May 3, 2012). 21 CFR 610.12
  2. ICH Q4B Annex 8(R1) Sterility Test General Chapter. U.S. FDA guidance for industry, September 2017. FDA docket FDA-2009-D-0013
  3. United States Pharmacopeia, General Chapter <71> Sterility Tests (as named in ICH Q4B Annex 8(R1), FDA guidance). USP <71>
  4. Parveen S, Kaur S, David SA, Kenney JL, McCormick WM, Gupta RK. Evaluation of growth based rapid microbiological methods for sterility testing of vaccines and other biological products. Vaccine. 2011;29(45):8012-8023. PMID 21871516 · DOI 10.1016/j.vaccine.2011.08.055
  5. U.S. FDA. Guidance for industry on aseptic processing and current good manufacturing practice (CDER, CBER, ORA), 2004. FDA docket FDA-2003-D-0145
  6. Bathgate H, Lazzari D, Cameron H, McKay D. The incubation period in sterility testing. J Parenter Sci Technol. 1993;47(5):254-257. PMID 8263662
  7. England MR, Stock F, Gebo JET, Frank KM, Lau AF. Comprehensive evaluation of compendial USP<71>, BacT/Alert Dual-T, and Bactec FX for detection of product sterility testing contaminants. J Clin Microbiol. 2019;57(2):e01548-18. PMID 30541938 · DOI 10.1128/JCM.01548-18 · PMC6355548
  8. Wallace NE, Kidwai AS, Knight M, Yang A, von Wintzingerode F. Combining a respiration-based method with universal qPCR for 3-day rapid sterility testing. PDA J Pharm Sci Technol. 2026 (online ahead of print). PMID 42601221 · DOI 10.5731/pdajpst.2026-000020.1
  9. IJzerman-Boon PC, Manju MA, van den Heuvel ER. Non-inferiority testing for qualitative microbiological methods: assessing and improving the approach in USP 1223. J Biopharm Stat. 2022;32(6):915-941. PMID 36131516 · DOI 10.1080/10543406.2022.2065498
  10. ICH Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products. U.S. FDA guidance for industry, August 1999. FDA docket FDA-1998-D-0003