By Miriam Guest, principal microbiologist, AstraZeneca
Confirmation of microbial quality of drug products manufactured by contract development and manufacturing organizations (CDMOs) is a key component of product release. The responsibility for product release lies with the CDMO’s quality organization, and the sponsor is required to ensure the appropriate quality of the article.
Microbial quality is not assured by end product testing alone; however, it is a critical release attribute. Compendial methods for sterility can be found in the harmonized pharmacopeias (USP, Ph. Eur., JP1,2,3). Within the framework of the compendial chapters there are a variety of approaches that can be made to meet the requirements.
The compendial methods provide a starting point to define a product-specific method, but the presence of the test article can lead to interference with the proliferation and detectability of microorganisms. It is essential the product-specific method is demonstrated to be suitable in the laboratory that is conducting the routine testing.
For sterility tests, two basic principles are applied. First, if a product is suitable for membrane filtration, this is the preferred methodology, as it reduces the potential for the product to interfere with the detection of microorganisms. In cases where material is not suitable for membrane filtration (for example, polymer injectable depots), direct inoculation may be performed.
For sterility, the methods across the three pharmacopeias are considered to be interchangeable.4
The execution of method qualification, suitability, and validation varies between laboratories. The reagents used, equipment, and level of experience can all impact the outcome of a microbiological method. Therefore, it is important to ensure that laboratory-specific method suitability testing is performed to ensure appropriate recovery of the presence of potential contaminants.
General Considerations For Microbial Test Methods
USP<1117> Best Microbiological Practice5 outlines the requirement for companies to have appropriate oversight of suppliers. Microbiology knowledge and experience are described as critical for adequate oversight of a supplier; thus, it is not sufficient to simply defer microbial laboratory testing to a third party, without the appropriate knowledge. An experienced microbiologist should initiate ongoing communications and serve as a technical expert.
To assure the suitability of all growth media used, quality control checks are performed prior to use; these are prescribed in the harmonized compendia. An area of difference between laboratories is the source of nutrient media; some facilities will prepare and sterilize their own media, typically from ready-made powder mixes, while others will purchase media that is ready to use. The quality control checks are important for both sources of media. In circumstances where media is prepared and sterilized at the site, the performance of the sterilization process may interfere with the media. For media that is purchased ready to use, the quality control checks performed at the testing site are to confirm that events during transportation have not impacted the performance of the media. The quality control checks include both sterility and fertility testing. In the case of fertility testing, the test panel of organisms is prescribed in the relevant pharmacopeial chapter. The sources of these organisms may vary; for example, cultures can be purchased, certified of origin from a known culture collection, or a laboratory may maintain a culture library at the site, which is used for such tests. The source of control organisms is also a factor to consider in method suitability testing.
General Considerations For Sterility Tests
There are two nutrient medias prescribed for sterility testing: Fluid Medium Thioglycolate for the detection of aerobic and anaerobic bacteria and tryptone soya broth for the detection of bacteria, yeasts, and molds.
Sterility testing should be performed in an isolator, with appropriately qualified decontamination cycles. Product-specific decontamination cycles may be required and an assessment of the potential ingress of vapor phase hydrogen peroxide should be made on a product-specific basis. Rapid transfer ports may be used in some isolator systems.
In some cases, the test article can render the nutrient media turbid, which makes the detection of microbial proliferation challenging. In those circumstances, a subculture step may be performed following the full 14-day incubation. Should a subculture step be required, it is important to minimize the risk of cross contamination during this activity, and it should typically be performed in a testing isolator.
During the incubation period, it is commonplace to inspect the nutrient media periodically throughout the prescribed incubation period. This inspection can vary between laboratories, in both the frequency of checks and whether a contemporaneous second check is performed at interim timepoints. It is best practice to conduct a contemporaneous second check at the completion of the incubation period.
The inspection of the media itself can also vary between laboratories, such as, for example, the adoption of standardized inspection lamps or the visual inspection in the laboratory setting.
Sterility By Membrane Filtration
Where a product can undergo membrane filtration, this is the preferred technique for sterility testing.
Typically, sterility test canister systems are purchased sterile and ready to use, and a number of vendors are available. These kits include a pair of sterility test canisters, with closed tubing systems. To access product, needles are typically in place at the ends of the tubing that can be used to pierce the test article primary package. The selection of the test canister system should consider the presentation of the test article and the product’s compatibility with it.
The material of construction for the filter membrane may be cellulose acetate or PVDF, and the selection of the appropriate membrane should consider any product-specific aspects.
A range of sterility test pumps is available that can be used with sterility test canisters from different suppliers. Pump speeds may vary, and it is important to understand that the product passes through the parallel filter membranes in the same volumes.
Rinse fluids are included in the harmonized pharmacopeia chapters; however, where products are inhibitory to microbial growth, additional neutralizers may be included if appropriately verified. In addition, neutralizers can be added to the nutrient broth or additional rinse fluid volume may be used to mitigate this.
Sterility By Direct Inoculation
Samples are directly inoculated into the nutrient medium prior to incubation. This approach is used where the test article cannot undergo membrane filtration, such as long-acting depots.
In these cases, consider whether the product could interfere with the ability to read the sterility test.
The volume of media may be adjusted to overcome product interference, and the use of neutralizers may be required to overcome inhibition of microbial growth.
There are a number of ways to meet the requirements outlined in the pharmacopeia sterility test chapters, and it is important to understand the method that will ultimately be used to support the microbial quality of the final product.
Laboratory-specific practices may influence the outcome of the result, and the test article may also interfere with the performance of the test. It is essential that each laboratory conducting the microbiological test on the test article undergoes product-specific method suitability to confirm method performance.
When establishing contracts with the test laboratory, additional factors to consider are the processes for laboratory investigations and out of specification results and clear expectations of timelines for communication. In addition, sampling, storage of samples, and retention periods should be factored in.
The location of sterility testing is another area that can influence the results of routine testing, and isolators are recommended for this. This minimizes the risk of cross contamination during testing, but the performance and qualification of such isolators should be considered. An understanding of the operations and product-specific method verification should be ascertained.
When working with CDMOs, it is important to build a relationship that facilitates open discussion and that ensures the sponsor has appropriate oversight of the testing that is being conducted on its behalf.
- United States Pharmacopeia (USP). The National Formulary, USP-NF 2022, Issue 1.
- European Pharmacopeia (Ph. Eur.), 11th Edition
- Japanese Pharmacopeia (JP), 18th Edition
- ICH guideline Q4B Annex 8 on evaluation and recommendation of pharmacopoeial texts for use in the ICH regions sterility test - general chapter 21 June 2017, EMA/CHMP/ICH/645592/2008, Committee for Human Medicinal Products
- USP<1117> Microbiology Laboratory Best Practices, United States Pharmacopeia
- USP<71> Sterility Tests
- Ph. Eur. 2.6.1: Sterility
- JP 4.06: Sterility Test
About The Author:
Miriam Guest is a principal microbiologist in Pharmaceutical Technology & Development, Global Operations, AstraZeneca, working in the New Modalities and Parenteral Development group, based at the company’s Macclesfield site in the U.K. She has worked in pharmaceutical development for more than 20 years, in both microbiology and formulation laboratories and GMP manufacturing environments. More recently, she has been supporting complex supply chains and designing robust control strategies, including the associated regulatory authoring and interactions. She leads the AZ Global Microbiology Forum, bringing together the global network of microbiology labs to drive standardization and quality principals in microbiology. Guest devised and leads AstraZeneca’s 21st Century Microbiology Strategy to innovate, industrialize, and implement technology solutions to drive efficiencies and process robustness benefits. She is an active committee member of the Pharmaceutical Microbiology Interest Group (Pharmig) and is involved in various cross-industry consortiums, including BioPhorum and the M3 Collaboration.