Current compendial methods for sterility testing in the pharmaceutical industry remain culture-based and include an incubation period of 14 days. Clearly, this is a delay that is becoming less and less acceptable in a modern manufacturing operation. However, there are signs that the situation is changing. For example, initiatives such as ‘Process Analytical Technology’ (PAT) and parametric product release are challenging the need for sterility tests to be completed before product can be released and both the FDA and the EMEA are encouraging the adoption of new analytical technologies to help ensure final product quality. In the USA the FDA Centre for Biologics Evaluation and Research (CBER) is proposing radical changes to the sterility test requirements for biological products, though not so far for all pharmaceuticals, promoting the use of rapid microbiological methods (RMM) as alternatives to the compendial sterility test methods. Such changes in the regulatory climate are creating renewed interest in RMM for sterility testing and several technologies are already available commercially.
ATP-bioluminescence:- Adenosine triphosphate (ATP) bioluminescence is a well established rapid method utilising a specific substrate and enzyme combination, luciferin/luciferase, to break down microbial ATP from growing cells and produce visible light, which can be measured using a luminometer. Several commercial systems have been developed for a range of pharmaceutical test applications, including sterility testing, especially for filterable samples where non-microbial ATP in the sample is less of a concern. The test time can be reduced considerably because detection of microbial growth in culture media is accomplished by ATP-bioluminescence, rather than by visible turbidity. Typically, results equivalent to those of compendial tests are available within 7 days or less. An example is the Celsis Rapid Detection System, combining the company’s Advance luminometer and AMPiScreen™ ATP-bioluminescence assay reagents, which use proprietary enzyme technology to increase the quantity of microbial ATP produced and reduce detection times by 25-50%.
The Milliflex® Rapid Microbiology Detection and Enumeration system from Millipore also uses ATP-bioluminescence to detect microbial cells and is designed specifically for monitoring microbial contamination in filterable samples. It is automated, employing image analysis technology to detect micro-colonies growing directly on the surface of a membrane filter after the addition of bioluminescence reagents. The system is designed to be quantitative, but a method has been developed and validated to use it for a rapid sterility test with an incubation time of just five days.
Colorimetric growth detection:- Colorimetric growth detection methods rely on a colour change being produced in a growth medium as a result of microbial metabolism during growth, often as a result of CO2 production. The best example of a commercial colorimetric assay system, which can be used for sterility testing is the BacT/ALERT® 3D Dual-T Microbial Detection System from bioMerieux. The system is automated and employs sensitive colour detection and analysis technology to produce a result in as little as three days. It can detect both aerobic and anaerobic bacteria, as well as yeasts and moulds.
Autofluorescence detection:- All living cells produce a small amount of fluorescence (autofluorescence) and this can be used to detect microbial colonies growing on a solid surface long before they are visible to the naked eye. This technique is particularly useful for filterable samples, where a membrane filter can be incubated on a conventional nutrient medium and scanned using highly sensitive imaging systems to detect microcolonies, sometimes several days earlier than using traditional colony counting methods.
Autofluorescence detection has been commercialised by Rapid Micro Biosystems as Growth Direct™, which uses a large area CCD imaging system without magnification to detect developing microcolonies. Although not yet validated for testing sterile products, “proof of concept” has been establish can be used for sterility testing is the BacT/ALERT® 3D Dual-T Microbial Detection System from bioMerieux. The system is automated and employs sensitive colour detection and analysis technology to produce a result in as little as three days. It can detect both aerobic and anaerobic bacteria, as well as yeasts and moulds.
Cytometry systems:- Cytometry does not rely on microbial growth to detect contamination, but instead uses cell labelling techniques to detect viable microorganisms. This approach has the potential to detect a wide range of organisms, including yeasts and moulds, within minutes. Commercial systems utilise combined fluorescent cell labelling and flow cytometry or solid phase cytometry to detect viable microbial cells. Typically, the cells are labelled using a fluorescent dye or a non-fluorescent substrate, which is converted to a fluorochrome in viable cells. Detection of the labelled cells occurs by laser scanning in either a flow cell (flow cytometry), or on a solid phase platform such as a membrane filter (solid phase cytometry). AES Chemunex has developed solid phase cytometry detection systems. The company’s Scan®RDI (also known as ChemScan RDI) system is capable of detecting 1 CFU per sample and has been evaluated as a possible RMM for sterility testing. The technology has been developed for the Stereal®-T sterility testing system.
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