Sterility Test Failure: Possible Causes and Investigation

Sterility Test Failure: Possible Causes and Investigation
Kerri Hirst, Microbiologist, ARL Bio Pharma

A sterility test detects microbial contamination and provides data to determine if your product is ready for release.  A “Sterile” result indicates no contaminating microorganism is found in the sample. A “Not Sterile” result indicates microbial growth and the product examined does not comply with the test for sterility, unless it is demonstrated that the test is invalid. An investigation is performed to determine if a sterility failure is invalid due to laboratory error. 

Sterility Test Failure (Out of Specification)

Once a sterility test is initiated, trained microbiologists periodically observe growth media for turbidity or fogginess indicating microbial growth. Microbial contamination may appear at any point during the incubation (Day 1-14) and subculture (Day 14-18) period.  Once contamination is confirmed, the test is out-of-specification (OOS) and an investigation is required to determine the cause of a failed sterility test. 

Possible Causes of Sterility Test Failure

  • compromised container integrity
  • improper aseptic processing and facilities
  • invalid sterilization processes
  • false positives

For more information on potential causes see USP chapters:

  • USP <71> Sterility Tests
  • USP <1207> Package Integrity Evaluation – Sterile Products
  • USP <1211> Sterility Assurance
  • USP <1229> Sterilization of Compendial Articles

ARL Bio Pharma’s Sterility OOS Investigation Process

An OOS investigation is conducted for every sterility test failure. This investigation consists of:

  • detailed examination of sterility test method
  • environmental monitoring data
  • sanitization logs for ISO 8, 7 and 5 environments
  • DNA sequencing reports
  • deviation reports (if applicable)
  • negative control data
  • potential retest results, if the test meets invalidation criteria

ARL Bio Pharma’s criteria for invalidating a sterility failure is a species level microbial identification match between ISO 5 environmental monitoring isolate(s) from the days surrounding the test date, or  evidence that the test was compromised due to sterility test controls or materials failures.  If there is a microbial identification match or failure of the test controls/materials, a sterility retest using the same lot, number of articles, and test method completes the criteria for invalidation.   

A match between an organism from the sample and ARL’s environment does not guarantee that contamination occurred during sterility testing, as common organisms may also be present in the original compounding environment.  While a species level match satisfies a portion of the invalidation requirements described in USP <71>, a sterility failure should prompt an investigation of compounding sterility assurance practices, equipment, and environmental monitoring at the facility in which the product was prepared. 

ARL has observed sterility failures due to multiple causes. In one instance, a “not sterile” result was invalidated due to a cracked filter container.  The growth media used to test a Vancomycin product for sterility was turbid at the 14 day observation point, and, upon further examination of the media container, a crack was discovered.  A discussion with the media container manufacturer confirmed this was a likely source of contamination.  A retest using the same number of articles was performed, using the same test method, and a sterile result was produced which invalidated the original OOS result.

While ARL investigates each sterility failure thoroughly, it does not have access to client data regarding sterility assurance practices, equipment qualification, sterilization validations, personnel training or environmental monitoring.  The only information ARL can utilize in investigating the cause of a sterility failure is internal data, which allows for determination of false positives or failure of test controls/materials. Any other sterility failure causes can be determined using data from the facility in which the product was compounded. Pharmacies and outsourcing facilities should assess all data to determine how to proceed following a sterility test failure.