The pharmaceutical and biotechnology industries face increasing pressure to comply with stringent regulatory standards, especially following the 2022 update to the EU GMP Annex 1 regulation.
This update mandates higher environmental monitoring (EM) standards, particularly in Grade A cleanrooms, where continuous air monitoring has become a requirement.
The adoption of RMMs, specifically BFPC technology, represents a significant shift from the well-established agar-based methods that are the long-standing reliable cornerstone of EM
As sterile product manufacturers adapt to these new standards, there is a growing interest in rapid microbiology methods (RMMs), such as biofluorescent particle counters (BFPCs), to complement traditional growth-based techniques.
The adoption of RMMs, specifically BFPC technology, represents a significant shift from the well-established agar-based methods that are the long-standing reliable cornerstone of EM.
This article will explore how BFPCs compare to traditional methods, the validation process required for implementing these advanced technologies, and the specific applications where BFPCs can provide significant advantages.
Traditional growth-based methods: A historical perspective
Agar-based methods are well-established in the detection of airborne microorganisms in cleanrooms. Plates, filled with nutrient-rich agar, are either placed in strategic locations within a cleanroom or used with active air samplers to assess contamination risk based on the presence of microorganisms from the environment. After an incubation period, the number of colony-forming units (CFUs) is counted to assess the microbial load in the area.
While settle plates and other growth-based methods are well-established and widely used, they have challenges. Mainly the time between sample collection and result analysis, typically requiring several days of incubation. This delay must be managed carefully to prevent any risk of contamination events escalating before appropriate corrective actions can be taken.
Traditional growth-based methods provide a snapshot of the microbial load at the time of sampling
Traditional growth-based methods provide a snapshot of the microbial load at the time of sampling. This approach can miss transient contamination events, which may occur between sampling intervals. Additionally, the accuracy of these methods can be influenced by factors, including the duration of exposure and cleanroom air flow patterns, which may not always provide a representative sample of the environment.
Traditional methods are also dependent on the ability of the sampled microorganisms to grow on the plates. Therefore, the selection of media types, incubation times and temperature can all have an impact on the effective growth of these microorganisms.
Stringent cleaning practices, utilising various disinfectant methods, could stress the microbial cells, negating their ability to grow. In such circumstances, the presence of viable but non-culturable (VNBC) bacteria cannot be detected using growth-based methods.
The rise of biofluorescent particle counters
Biofluorescent particle counters (BFPCs) offer a modern alternative to traditional growth-based methods. BFPCs, such as the Bio-Aerosol Monitoring System (BAMS) (Figure 1), detect, quantify, and size airborne biological particles in real-time, by measuring the fluorescence emitted by metabolites within microorganisms.
By delivering real-time results, BFPCs enable immediate detection of contamination events. This rapid response capability can significantly reduce the time to remediation, thereby minimising the risk of product loss and ensuring the integrity of sterile manufacturing processes.
Validation is crucial when comparing BFPCs to traditional methods, as regulatory bodies expect new technologies to provide results that are at least equivalent, if not superior, to established practices
Additionally, BFPCs are capable of detecting VBNC microorganisms that would not be identified by traditional, agar-based methods.
Despite these advantages, the implementation of BFPCs requires careful validation to ensure that the technology meets the specific needs of the end user.
Validation is crucial when comparing BFPCs to traditional methods, as regulatory bodies expect new technologies to provide results that are at least equivalent, if not superior, to established practices.
Validating BFPC Technology: A structured approach
Validation of BFPC technology involves a series of steps designed to demonstrate that the instrument is fit for purpose and can produce reliable, accurate results for a specific application. This process typically follows a framework that includes Installation Qualification (IQ), Operational Qualification (OQ), and Performance Qualification (PQ).
Installation and Operational Qualification (IQ/OQ)
IQ/OQ phases ensure that the BFPC is installed correctly and operates as intended. During IQ, the installation of the BFPC is documented, including verifying that all components are present, correctly configured, and compliant with design specifications.
IQ/OQ phases ensure that the BFPC is installed correctly and operates as intended
OQ focuses on confirming that the BFPC performs according to its operational specifications in the user’s environment. This involves verifying that the instrument operates correctly under actual conditions, including performance, calibration, and functionality checks. It also includes ensuring data accuracy, software integration, and compliance with relevant standards, all documented thoroughly.
Performance Qualification (PQ)
PQ is the most critical validation phase, where the BFPC's performance is tested under actual operational conditions. PQ is user-defined and tailored to the specific application of the BFPC. It consists of three primary components: equivalence testing, accuracy and precision calculation, and interferent testing.
Equivalence testing: This is essential when implementing BFPC technology alongside or as a replacement for traditional growth-based methods. The goal is to demonstrate that the BFPC can provide comparable results, ensuring that critical quality decisions can be made with the same level of confidence.
In practice, equivalence testing involves side-by-side comparisons of BFPC readings with those obtained from the traditional method in various cleanroom environments.
The goal is to demonstrate that the BFPC can provide comparable results, ensuring that critical quality decisions can be made with the same level of confidence
For example, in a Grade A cleanroom, where zero microbial counts are expected, equivalence testing may represent a challenge. It may be necessary to conduct testing in environments with greater allowance for microbial loads, such as lower-grade cleanrooms or simulated environments, to establish a baseline for comparison.
The BFPC's alert and action levels are then calibrated to match those of the traditional method, ensuring that the BFPC can reliably detect contamination events that would trigger a response under the established protocols.
Accuracy and precision calculation: The data gathered during equivalence testing is used to calculate the accuracy and precision of the BFPC.
Accuracy refers to the BFPC's ability to correctly count the number of biologic particles in a sample compared to the traditional method. For example, if settle plates record 100 CFUs per cubic metre, the BFPC should report a similar count, typically ≥70% of the traditional method's result. This threshold ensures that the BFPC provides a reliable indication of microbial load.
Precision measures the repeatability of the BFPC's results
Precision measures the repeatability of the BFPC's results. Multiple samples are taken under identical conditions, and the variation in results analysed. The BFPC should demonstrate consistent results with low variability, comparable to the precision of the traditional method.
These calculations are crucial in establishing confidence in the BFPC's ability to accurately monitor the cleanroom environment, ensuring that it can be relied upon for ongoing EM. However, the specific application of the BFPC may result in more or less stringent testing.
Interferent testing: This assesses the BFPC's ability to distinguish between biological and non-biological particles. It is essential to ensure the BFPC does not produce false-positive results due to the presence of non-microbial materials that may fluoresce under the instrument's detection wavelength, such as certain plastics or fibres.
The challenge with agar-based is mainly the time between sample collection and result analysis
In this phase, materials commonly found in the cleanroom are introduced to the BFPC under controlled conditions to determine if they cause interference. If false positives are detected, alternative materials should be considered, or the use of these materials should be restricted during active monitoring periods.
Application-specific validation: Tailoring BFPC Use
The versatility of BFPC technology allows it to be applied in a variety of cleanroom environments and applications. This flexibility necessitates a tailored validation approach depending on the specific use.
The following are examples of how BFPCs such as BAMS can be validated for different applications:
1. BFPC in Isolators
Isolators used in sterile manufacturing are designed to maintain a Grade A environment with minimal particle counts. Validating BFPCs in such environments is challenging due to the expected low microbial load.
Biofluorescent particle counters (BFPCs) enable immediate detection of contamination events
To address this, BFPCs should be tested in different environments, ranging from high-level cleanrooms to more contaminated areas, to establish their accuracy and precision across a range of conditions.
Additionally, materials used within the isolator should undergo interferent testing to ensure they do not contribute to false-positive readings.
2. BFPC for HVAC system investigations
BFPCs can be invaluable in investigating the effectiveness of HVAC systems, such as detecting leaks in HEPA filters or monitoring air exchange rates.
PQ is the most critical validation phase
Validation in this context would focus on the BFPC's ability to detect changes in particle counts as the system's performance is adjusted. The accuracy and precision of the BFPC in detecting changes are critical for ensuring that the HVAC system maintains the required air quality standards.
3. Investigational Studies
BFPCs can also be used for investigational purposes, such as studying the impact of specific behaviours or practices on cleanroom contamination levels.
BFPCs can also be used for investigational purposes, such as studying the impact of specific behaviours or practices on cleanroom contamination levels
For example, an aseptic technique study could involve monitoring particle counts as personnel perform different tasks. Validation would involve ensuring that the BFPC can detect subtle changes in particle levels and provide consistent, repeatable data.
Ensuring compliance and enhancing contamination control
The implementation of BFPCs represents a significant advancement in environmental monitoring for sterile manufacturing. These rapid microbiology methods offer real-time, continuous monitoring, providing a more comprehensive and responsive approach to contamination control than traditional growth-based methods like settle plates.
By following a structured validation approach that includes equivalence testing, accuracy and precision calculations, and interferent testing, manufacturers can confidently integrate BFPCs into their environmental monitoring programmes
However, the adoption of BFPC technology requires a rigorous validation process to ensure that it meets the specific needs of each application.
By following a structured validation approach that includes equivalence testing, accuracy and precision calculations, and interferent testing, manufacturers can confidently integrate BFPCs into their environmental monitoring programmes. Therefore, this technology will play an increasingly important role in the evolving pharmaceutical industry.
