Scientific progress in the field of cellular and molecular biotechnology has led to a fast development of biopharmaceuticals, tissue engineered applications and advanced therapy medicinal products (ATMPs).
The safety of such novel technologies, products and applications is very important. One area for risk assessment and safety is the detection, prevention and control of contamination from microorganisms or mycoplasmas.
Many modern biopharmaceutical products are based on large molecules, cells or tissues that have a complex structure. Thus the classic compendial microbiological test methods are often not suitable for these substances and products.
Additionally, many modern therapeutics have a short shelf-life. Therefore, it is essential to receive results well before the end of their shelf-life.
Meanwhile, numerous scientists and companies have been working on the development of suitable rapid alternative microbiological methods for the faster detection and identification of microorganisms. As a result, various systems with different methodologies have been launched: PCR based, MALDI-ToF Mass Spectrometry, flow cytometry, etc.
Oliver Gordon et al. (2011)1 outlined in an Overview of rapid microbiological methods (RMMs) evaluated, validated and implemented for microbiological quality control: “RMMs hold a lot of potential to accelerate and even improve microbiological quality control. Besides allowing drug products to reach the market and therefore the patient earlier, RMMs also allow quick and effective investigative and corrective actions in case of a microbiological quality issue. The growing interest for RMMs leads to an increase of different systems available from different suppliers, offering choice to identify the most suitable system for the intended application.”
Development targets for such new technologies are increased accuracy, reproducibility, sensitivity, with (possibly) automated, miniaturised and high-throughput processing. Automation, in particular (e.g., with a LIMS system), allows for optimised data handling as well as easier analysis and the ability to follow trending of microbiological data.
Depending on the target, it is principally possible to divide the alternative method into quantitative methods, qualitative methods and in methods for microbiological identification.
Following developments in science and industry many related regulatory documents were recently revised. These include: Ph. Eur. chapter 5.1.6. “Alternative Methods for Control of Microbial Quality” or USP chapter <1223> “Validation of Alternative Microbiological Methods” as well as state-of-the-art guidelines such as the PDA Technical Report 33 “Evaluation, Validation and Implementation of New Microbiological Testing Methods”.
A look into the currently published Ph. Eur. chapter 5.1.6. revision shows that it includes the following content:
- Basic principles for detection, enumeration, isolation and identification of the methods that have successfully been used in the QC of pharmaceuticals
- Guidance on validation of alternative methods
- Equivalence testing against compendial methods
After the evaluation of the user requirement specification (URS) and the Design Qualification, the validation for the intended use of new methods goes on with the classical steps of qualification of the equipment/instrumentation and the associated software/computer system (IQ/OQ).
Installation Qualification (IQ) – in this case means establishing by objective evidence that all key aspects of the process equipment and ancillary system installation adhere to the approved specification. It also means that the recommendations of the supplier of the equipment are suitably considered. Operational Qualification (OQ) – is the compilation of pragmatic evidence that a process can consistently produce regulatory controlled product within predetermined specifications.
These steps are followed by the validation of the test method – the Performance Qualification (PQ) step. Here it is necessary to show that with standardised microorganisms in known concentration in a suitable diluent the selected method does meet specific acceptance criteria and/or is equivalent or non-inferior to the currently given compendial test method.
The choice of a new microbiological test method is particularly important for the suitability and successful implementation. The essential points that need to be considered here are:
- Is the method destructive; so identification may not be possible (some presence/absence methods)?
- Stressed microorganisms – is the detection of non-viable or viable but non-cultivable micro-organisms of concern?
- Can pure colonies be found?
- Will the micro-organisms be present in the reference database used?
- The limitations of both the conventional and alternative method
After the above mentioned validation of a selected method, its suitability for its current use must be proven. To this end, the test must show that the test sample does not interfere with the technology’s detection capacity or microbial recovery. It further has to define the acceptance criteria in terms of application and validation data. Additionally, equivalence testing is needed to show the comparability of the alternative and the compendial method.
Equivalence testing requires sufficient numbers of replicates for relevant strains of test micro-organisms and a parallel testing which is justified by a risk assessment and requires parallel testing of samples for a pre-specified period of time. The revised Ph. Eur. chapter 5.1.6. expects a qualitative method to use the same pass/fail result as for a compendial method. For a quantitative method, a statistical analysis must be at least equivalent to the compendial method.
Not only have important parts of the European Pharmacopoeia been revised but the relevant chapters of the US Pharmacopoeia were updated as well as the PDA TR 33. Dr Michael Miller presented tables 1 and 2 comparing the validation procedures in these three documents at PharmaLab 2016.
Independent of what method is used and whether we talk about suitability or equivalence testing, all decisions should be based on a scientific rationale. This rationale must be substantiated by a relevant number of test data. That means that the obtained data must be collected and analysed in a suitable manner. Therefore statistical methods form an indispensable tool to quantify the performance. Experience from past years shows that close cooperation of the responsible microbiologist and statistical experts facilitate the analysis and interpretation of such microbiological data during the method validation phase. Subsequently this strengthens the process of method validation and makes it easier to develop the scientific rationale. And this rationale is needed for the implementation of alternative microbiological methods and to justify the switch to a non-compendial method for the responsible authority.
The validation, especially of modern microbiological methods, and the use of statistics methods, including the tools, will be at the focus of the ECA special workshop Validation of Microbiological Methods on 11-12 May 2017, in Prague, Czech Republic. See www.microbiology-conference.org for more information.