Regulatory Standards for Sterility Testing

Biologics Characterization

Feb 22, 2021 | 3 min read

What is Sterility Testing?

During the production of biopharmaceuticals, contaminating microorganisms can enter the process at many stages. Once introduced, bacteria or fungi can replicate exponentially, severely compromising the final product. The presence of microorganisms in vaccines and other biopharmaceuticals can over time both spoil the product and pose a major risk to patient health. As such, regulatory bodies including the FDA (1) and EMA (2) have stringent tests in place to ensure contamination has not occurred. This is determined through sterility testing of representative samples of a batch, to identify the presence of any viable microorganisms in the material. The proof of sterility is part of documentation required for the lot release of biopharmaceuticals.

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How Does Contamination Occur?

There are numerous sources of contamination during the production of biopharmaceuticals. This includes raw materials, personnel, equipment and the manufacturing environment. Regulatory agencies provide guidance for reducing the risks of introducing bacterial or fungal contamination, including the use of sterilizing techniques on raw material, ensuring aseptic conditions and through implementing GMP. However despite these precautions, it is not possible to eliminate risk entirely. As such, it is necessary to regularly test for sterility during manufacture.

Regulations for Determining Sterility

The guidelines for sterility testing are present in various pharmacopoeias worldwide, including the United States Pharmacopeia (USP) and the European Pharmacopeia (EP). This guidance has been harmonized by the IHC (3). Sterility testing performed by Sartorius is to GMP regulations and in compliance with the EP (2.6.1. Sterility) and USP (<71> Sterility tests).

Methods for Sterility Testing

Determining sterility involves the use of test material to inoculate media which supports microbial growth. Following inoculation, the media is monitored for 14 days for visual signs of bacterial or fungal contamination. If growth is observed, this can be confirmed using agar plates and the microorganism identified. This may help understand how the product was compromised.

The guidelines detail testing for sterility through inoculation of both fluid thioglycollate medium and soya-bean casein digest medium. A combination of these two media has been selected to support rapid growth of a wide-range of microorganisms, including both aerobic and anaerobic bacteria and fungi. Test material spiked at low levels with representative organisms (such as the aerobic bacterium Staphylococcus aureus, spore forming bacterium Bacillus subtilis, anaerobic bacterium Clostridium sporogenes or the fungus Candida albicans) are used to ensure there is no inhibitory effect from the sample which may mask contaminants, in order to validate the test.

Sterility testing can be performed using one of two methods, membrane filtration or direct inoculation. For direct inoculation, the test material is simply aseptically added to inoculate the media. For membrane filtration, the test material is passed through a size exclusion membrane capable of retaining microorganisms. The membrane can then be aseptically transferred into the medium. The membrane filtration method is recommended for accommodating large volumes of test material or when the test material contains substances which may inhibit growth of microorganisms, such as antibiotics.

How Much Material is Required for Testing?

The amount of material and the minimal number of samples to test is dependent on the number and size of the vials produced from each manufacture batch. The IHC describes the minimum number of items to be tested to confirm the sterility of the whole batch. At Sartorius, we can provide advice on the number of samples required for lot release.

Bioburden Testing

The need for sterility can vary between biopharmaceuticals. Some products, such as those intended for intravenous injection must be shown to be sterile, whilst other products may have a microbial limit set. This is the limit of microorganisms which the final product can contain so that risk to the consumer is extremely low but which is reasonable for manufacture. In these cases a bioburden test should be used as opposed to a sterility test. Bioburden tests provides an evaluation of the microbial content of a product. For this test, the sample is used to inoculate agar plates. These plates are incubated and the number of microbial colonies counted. This can be used to determine the overall number of microbes present in the product, which can be compared to the acceptable limits dictated by regulatory guidelines.

Conclusion

Testing the sterility of biopharmaceuticals is essential to ensure safety. If not identified, contaminants may result in life-threatening infections in patients. There are many examples of past incidences where products have been recalled due to the lack of sterility assurance (4). As such it is important to be confident that the testing procedures being performed are to regulatory guidelines. At Sartorius we can ensure that the required sterility testing is performed at GMP standards by dedicated expert biosafety scientists, providing you with the evidence needed for the lot release of your biopharmaceutical.