Sterility testing of final products is a mandatory release test for all drug products purported to be sterile required by Good Manufacturing Practices (c-GMP) and described in the European Pharmacopeia (EP) and the United States Pharmacopeia (USP) (1,2). Microbiological contamination can lead to recalls, compromise patient safety and impact a manufacturer’s reputation.
Due to lengthy time to result of traditional sterility testing methods (14 days), interest in more rapid methods for sterility testing is growing among pharma/biopharmaceutical manufacturers and quality control laboratories supporting these industries. Rapid sterility testing can enable faster time to market, reduced storage time, optimized production quality and batch processing, all of which can lead to significant cost savings and competitive advantage.
In many geographies, regulatory and industry guidelines encourage validation and adoption of rapid microbiology methods. The EP has created a dedicated reference (5.1.6) entitled “Alternative Methods for Control of Microbiological Quality” which provides guidelines for rapid methods including rapid sterility testing (3). The USP did the same with chapter <1223> “Validation of alternative microbiological methods” (4). Similarly, the Parenteral Drug Association (PDA) includes rapid sterility testing in its Technical Report 33 “Evaluation, Validation and Implementation of Alternative and Rapid Microbiological Methods” (5).
While guidelines are evolving and the need for a faster approach to sterility testing is well-recognized, few systems have been validated and implemented and then approved by regulatory authorities. This has slowed general acceptance and consequently adoption. This article describes the evaluation and validation process of a rapid sterility testing method which can deliver results in five days rather than fourteen.
Traditional Methods
The two traditional methods for sterility testing described by the EP and USP (1,2) are membrane filtration and direct inoculation. Membrane filtration is to be chosen whenever the product is filterable while direct inoculation is used when the product cannot be filtered. Both methods are based on the capacity of contaminants to grow, proliferate and become visible in an incubation medium. They require two liquid culture media: Fluid Soybean Casein Digest Medium (Trypticase Soy Broth = TSB) and Thioglycollate Medium (FTM), which are meant to allow recovery of all types of microorganisms normally present in a pharmaceutical environment, including aerobic and anaerobic bacteria, yeasts and molds.
Among the major disadvantages of traditional sterility testing methods is the subjectivity of the visual examination of the results. Because turbidity must be visually verified by laboratory personnel, the methods are subject to increased risk of human error.
Another disadvantage is that traditional sterility testing methods require at least 14 days to complete. During this time, companies incur costs to hold their products in storage until sterility is proven. Additionally, in case of test failure (growth), a corrective action to the process may be not performed quickly enough, thus compromising the quality of the future product batches.
Identifying a New Method
As a contract research organization which offers microbiological testing, Confarma partners with pharmaceutical and biopharmaceutical companies to ensure the sterility of raw materials, products for in-process controls and final drug products. For all sterility testing, Confarma adheres to global quality standards including Good Manufacturing Practices (c-GMP), Good Laboratory Practices (GLP), ISO 9001, ISO 17025, ISO 14001 and OHSAS 18001.
Confarma performs sterility testing for clients using the membrane filtration and direct inoculation methods. However, Confarma sought to provide an alternative method with faster results to enable its clients to release products sooner and, in the case of a non-sterile product, to start an investigation earlier.
The Confarma team researched several options for rapid sterility testing and identified four major criteria that were required for the new system:
•The alternative method needed to be similar to the traditional test to facilitate data interpretation and method validation
•The test needed to be performed in an isolator to reduce the risk of false positives
•In case of positive result (contamination), the method needed to be compatible to the available identification methods for further investigation
•For ensured quality of performance, the system needed to have been studied by the regulatory authorities previous to Confarma implementation
Of the systems researched, the Milliflex Rapid system (Merck Millipore, Darmstadt, Germany) was the only one which met all the criteria. Researched literature included articles written by Novartis (Basel, Switzerland) describing the validity of the system application and its regulatory acceptance from different authorities including the EMA and FDA (6,7,8). In an additional independent study, the Federal Drug Administration (FDA) Center for Biologics Evaluation and Research (CBER) confirmed the method to be acceptable as an alternate sterility method in comparison to other rapid systems (9).
It is important to note that there are a number of challenges associated with validating a new method. Implementing a new approach in an existing laboratory can be difficult. There is a learning curve associated with familiarity on new equipment and the laboratory may have to be redesigned to accommodate the new approach. When working with different requirements (EP/USP/PDA) there is varying information needed to comply with each; this necessitates additional research and organization to ensure compliance across all regulations. Additionally, alternative method validation demands generation of supporting statistics to verify that the results are compliant with specifications and to conclude whether the method is equivalent or superior to the traditional method. Confarma kept each of these challenges in mind to perform validation in an effort to streamline the process.
Validation
Once the Milliflex Rapid system was identified as the chosen method, Confarma began the validation process. Since Confarma has developed and validated several rapid methods in microbiology including mycoplasma detection by qPCR, a set of best practices, which are considered before starting the process, were in place:
•Regulations guiding the method should be studied to ensure that all requirements are met by the preferred method
•Regulations guiding the method should be discussed with the authorities to make sure all involved parties are in agreement about the proper regulations and procedures
•Other needs specific to the method should be evaluated; in this case a good example is the need for a statistician
•It is extremely important to have a multidisciplinary team to support the validation process.
For validation of the Milliflex Rapid system, the team consisted of the following roles:
•Microbiologist: responsible for validation design, issuing the protocol and results and overall project management and decision making
•Technician: performs manipulations and technical review of the protocol and results
•Statistician: responsible for analyzing the results, providing statistics and recommending a conclusion
•Quality assurance: in charge of reviewing the protocol and the results to verify that the regulatory requirements were satisfied and approbation of the final documents
•Responsible pharmacist: provides overall review and approval of the project
Confarma’s validation process was streamlined due to the detailed validation protocol provided by the supplier (Merck Millipore). Merck Millipore partnered with Confarma from feasibility studies throughout the entire validation process and helped navigate the transition from primary validation through qualification of installation (IQ) and operational qualification (OQ) as well as providing performance qualification (PQ) consultancy.
