Bacteriostasis and Fungistasis Testing: Sterility Validation

By Andy Whittard

Sterility testing and method validation have made the mainstream news lately, courtesy of the criminal court cases against the New England Compounding Center (USA). The NECC has been closed over its role in releasing contaminated methylprednisolone, a drug used for epidural steroid injections to relieve back pain. They were also found guilty of operating as an unlicensed manufacturer and the company leaders have been jailed.

Compounding pharmacies produce medications from scratch to fill individual prescriptions, so they are not required to test for sterility. They are regulated under US State Governance, as opposed to licensed manufacturers who must follow FDA regulations. The NECC failed to perform regular sterility testing. It was also not preparing the product with preservatives to inhibit bacterial and fungal growth.

Emails shown as evidence in the NECC trial revealed that the company head knew they should have been performing sterility testing all along. That was just weeks before it was discovered they had shipped out a contaminated product, across the United States. The resulting use of product contaminated with fungal meningitis killed over 50 people and injured almost 800 more.

What is sterility testing and method suitability?

Sterility testing is the series of assessments performed at every step through the sterile manufacturing process of products. These tests are designed to prove the absence of microbial contamination, and that the products will be sterile when they reach the consumer.

When the product contains a preservative, the sterility test must neutralise or dilute any antimicrobial activity. This ensures that the product is sterile due to your manufacturing processes, not due to the antimicrobial agents in the product.

Method suitability testing checks that microbial growth is not prevented from being seen under non-sterile conditions. This is required to assess appropriate sterility testing is performed and is crucial when preservatives are present in the product.

Bacteriostasis and fungistasis testing is a requirement by all major pharmacopoeias

Antimicrobial activity is part of the suitability testing, in that you are confirming the antimicrobial agents in the product have been neutralised. Without this confirmation, you cannot be sure if your sterility test shows that your manufacturing process is sterile. Contamination could be masked by the active antimicrobial agents in the product.

The US, European, and Japanese pharmacopoeias all require sterility testing, including method suitability testing. As the New England Compounding Center case shows, it is a crucial step in pharmaceutical manufacture. The process requires 14 days to produce a negative result and must be performed before the release of a product. This can be a burden to small-scale producers because it requires testing at least 2% of the output.

Along with the sterility testing, method validation is required for every new product and change in experimental conditions of the test. Once validated, if there are no changes, the Quality Risk Management must identify when to re-assess the method validation.

Make sure your validation test forms a part of your procedure

Any standard testing procedure needs to include positive and negative controls. If you do not know whether your testing procedure is capable of detecting contamination, how will you confirm sterility?

A positive sterility test can be caused by actual contamination or the test itself. If you don’t validate your testing processes properly you could waste valuable resources hunting for a contamination source that doesn’t exist. These controls pay for themselves when they detect inconsistencies.

Demonstrating the absence of antimicrobial properties that inhibit microbial growth

The pharmacopoeias set out very clear guidelines on how method validation is to be performed. There are two major types of tests: membrane filtration and direct inoculation.

Membrane filtration uses membrane filters with pores 0.45μm or smaller. This is designed to retain microbes during repeated rinsing steps to remove any preservatives, then tests for growth in suitable media. Ensuring that the media used captures both aerobic and anaerobic microorganisms.

Direct inoculation places the product directly in the suitable media for aerobic or anaerobic growth. It uses dilution or neutralisation to mitigate the effects of any antimicrobial compounds in the product. It is up to the manufacturer to decide which method is more appropriate.

The goal is to see if fungi or bacteria will grow in the product once all antifungal agents have been neutralised. Correct choices in neutralising agents - as part of rinses or those integrated into media - are crucial. Any method validation testing relies on consistently reliable products to reduce the chance of introducing uncertainty.

The media needs to be sterile before inoculation, and the appropriate components used to filter out or neutralise the antimicrobial compounds. This demonstrates the sterility test has the best possible chance of detecting any contamination, and eliminating factors that could produce a false negative result.

The New England Compounding Center case is a sobering reminder of the importance of performing sterility testing and method validation testing. By deciding to ignore validation procedures along with other short falls, the New England Compounding Center became responsible for the deaths and critical illness of many people. This was an extreme case of poor practice and neglect. Yet it demonstrates very strongly the consequences of operating without due testing procedures.

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