Sterility testing is a process which must be performed as part of the manufacture of sterile products, to provide confidence that they are free of any viable microorganisms which could harm patients. As it is not possible to test every single vial or ampoule of product that is being manufactured, a number of samples representative of the whole batch are taken at different times during the filling operation and tested for microbial contamination.

The world health organisation adopted requirements for sterility testing in 1973, and today the guidelines for conducting sterility testing are present in various pharmacopoeias worldwide, including the United States Pharmacopeia (USP) and the European Pharmacopeia (EP).

Insourcing vs Outsourcing

When conducting this mandatory process, manufacturers of sterile products have two options. Either they can perform the process in house inside a dedicated sterility testing lab or outsource process to a third party testing service.

Outsourcing is typically more beneficial for smaller manufacturers, as setting up a sterility testing lab can have a high initial cost, and also high running costs.

However, there are downsides to outsourcing such as longer wait times for test results leading to increased storage costs, particularly for products which require cold storage. Also, many testing services charge per test, which can become very expensive, particularly for larger manufacturers who manufacture a lot of product, so require a large number of tests.

Finally, outsourcing sterility testing to a third party means there is less control over the process, which can ultimately result in unnecessary scrapping of viable product, which will be explained further on in this article.

Methods

There are two methods for testing the sterility of products – membrane filtration and direct inoculation. By far the most common method is membrane filtration, which involves passing the liquid product through two canisters, each containing a filter capable of retaining viable microorganisms, then filling each of the canisters with different types of growth media – one for growing aerobic organisms, and the other for anaerobic organisms. By contrast, direct inoculation involves placing the product directly into the two canisters, and is typically used for products that cannot be filtered, such as medical devices. With both methods, the canisters will then be incubated at the appropriate temperature for 14 days, and if there are no signs of growth after this time, the test is considered a pass and the product can be released for delivery to patients. If either canister shows signs of growth (turbidity) then the test is considered a fail.

Test Failure

The impact of a test failure can be significant for a pharmaceutical manufacturer as it typically results in:

• Withholding release and potentially scrapping the batch of product which failed the test
• Regulatory involvement
• Performing timely investigations
• Halting production of further product whilst investigations are performed
• Additional cleaning and disinfection of production areas

Ultimately a sterility test failure will result in financial losses to the manufacturer, and potentially drug shortages to patients. A genuine failure prevents contaminated product from being administered to and potentially harming a patient.

However, sterility test failures are not always caused by contamination in the product.

False Positives

A false positive result is when contamination from a source extrinsic to the product, such as the environment or the operator performing the test, finds its way into the test and causes a failure. This will suggest that the product is contaminated when potentially it is free of microorganisms. In the case of a sterility test failure, the burden of proof is on the manufacturer to demonstrate that the failure is the result of contamination from the operator and/or lab environment. In reality, it is very difficult to prove this so a false positive result often leads to perfectly safe and effective products being scrapped unnecessarily.

Environment

To reduce the risk of false positives, the sterility testing process should be performed in an aseptic environment. Historically, the common approach to achieve this was by performing the process in a biological safety cabinet (BSC) or laminar air flow (LAF) hood. However, this means the process is still open to the environment and operator, and thus there is still a risk of false positives occurring.

More recently, the industry is shifting towards the use of isolators which provide a physical barrier between the operator/environment and the test, substantially reducing the risk of false positive results.

It is important to note that regulators and pharmaceutical advisory committees around the world have requirements and recommendations concerning the environment in which sterility testing should be conducted.

Regulations

EU GMP Annex 1: Manufacture of Sterile Medicinal Products section 10.6 states “The sterility test should be performed under aseptic conditions.” The FDA’s Guidance for Industry for Sterile Drug Products Produced by Aseptic Processing takes things one step further and states in section XI “The use of isolators for sterility testing minimizes the chance of a false positive test result.”

Furthermore, the pharmaceutical inspection co-operation scheme (PIC/S) provides an entire guidance document (PI 014-3) on isolators used for aseptic processing and sterility testing.

So, although there is no hard requirement to perform sterility testing in isolators, there is a strong benefit to do so in order to reduce the risk of false positives, which can result in financial losses to manufacturers of products, and drug shortages to patients.

What is An Isolator?

According to EU GMP Annex 1, an isolator is “An enclosure capable of being subject to reproducible interior bio-decontamination, with an internal work zone meeting Grade A conditions that provides uncompromised, continuous isolation of its interior from the external environment”. Isolators have several features to maintain Grade A conditions and isolation from the external environment such as:

• Airtight / inflatable seals on all doors
• Interlocks to prevent doors being opened once aseptic conditions have been achieved (following bio-decontamination)
• Unidirectional airflow between 0.36-0.45 m/s (to comply with EU GMP Annex 1 guidance values)
• HEPA filters to cleanup incoming air
• Positive pressure
• Leak/pressure testing of enclosure and gloves separately
• Environmental monitoring systems to confirm that the environment remains aseptic during use
• Airflow and pressure alarms
• Hydrogen peroxide bio-decontamination systems to help inactivate microorganisms on the enclosure surfaces and incoming materials needed for the testing process

Benefits of Isolators

Primarily, isolators reduce the risk of false positives occurring during sterility testing which can save manufacturers of sterile products millions of dollars by minimising unnecessary scrapping of product. As a secondary benefit, isolators can also provide substantial savings through operating costs. Unlike a BSC/LAF which must be situated in a Grade B cleanroom, sterility test isolators can be situated in a lower Grade D cleanroom as they are isolated from their surrounding environment. This can result in substantial savings from:

• Reduced energy bills due to lower capacity HVAC system
• Reduced cleaning and disinfection consumable costs
• Reduced labour resource (for cleaning and disinfection)
• Less gowning
• Less maintenance of the cleanroom
• Increased efficiency of operators as lower gowning means they can work for longer periods of time

In fact, one study¹ found that isolators can reduce cleanroom running costs by up to 72%. Despite the above benefits, it is important however to note that isolators do have some downsides in comparison to open LAF/BSC environments:

• They typically have a higher up front / equipment cost. However, this can be offset by the savings from lowering the cleanroom grade and reduced risk of scrapping of product.
• Operators must perform their duties through gloves connected to sleeves, which can be more difficult than gloved hands operating inside a BSC/LAF, so can lead to less efficient work, slowing down a process. However, operators do not have to wear such restrictive gowning in grade D cleanrooms, meaning they can work for longer periods of time in more comfortable conditions.
• Finally, although automated bio-decontamination of an isolator requires less operator workload than manually disinfecting items into a BSC/LAF, the loading and bio-decontamination process does typically take longer, in some cases taking hours, which can add substantial time to the testing process or requires adjustment to working patterns by loading and bio-decontaminating the isolator overnight ready to start the testing process in the morning. However, there is a way to overcome this issue of long cycle times...

Modular Isolators

One of the biggest hurdles to overcome when switching from a BSC/LAF to an isolator is to maintain the same throughput i.e. number of tests, due to the long bio-decontamination cycle. But Ecolab’s Bioquell Qube isolator solves this problem by adopting a modular approach to isolator systems. Rather than having one large isolator chamber, which is filled with all required materials needed for a full day of testing and subsequently running a long bio-decontamination cycle, the Bioquell Qube utilizes a multiple chamber principle whereby one small chamber is filled with a small amount of materials to do several sterility tests, and bio-decontaminated. Then these materials are transferred into an adjacent chamber where the pump resides, the interconnecting door is closed, and whilst the testing is being conducted in one chamber, the materials required for the next tests are bio-decontaminated in the adjacent chamber. With a small bio-decontamination chamber and a relatively light material load, bio-decontamination cycle times of as little as 30 minutes can be achieved (subject to quantity and absorbency of load).

¹Costing a cleanroom per square foot, Cleanroom Technology, 28 February 2018, https://www.cleanroomtechnology.com/news/article_page/Costing_a cleanroom_per_square_foot/139470