Microbial characterisation solves sterility issues
Pharmaceutical companies are relying increasingly on genotypic methods for microbial contaminant identifications. Colin Booth, vice-president, science and technology at Oxoid, describes a ribotyping method that offers microbial characterisation in addition to rapid species identification
It is extremely important for pharmaceuticals, and other health-related products, to be free from micro-organisms that might be harmful to the individuals taking or using them. When an unknown microbial contaminant appears in a pharmaceutical product, production may have to cease until the problem is identified and rectified.1 The longer manufacturing is at a standstill, the more revenue is lost and, if the product is in development stages, clinical trials may have to be halted, resulting in delays in bringing the product to the market.
Knowing and understanding the microb;ial make-up of the manufacturing environment can help to avoid such sterility problems - but, if and when they do occur, identifying the problem, locating the source and putting it right as quickly as possible can salvage what has already been invested in the product.
Genotypic vs Phenotypic methods
Phenotypic methods for microbial identification are widely available and used in a variety of applications. Many of these methods, however, are complicated, labour intensive, and interpretation of the result may be subjective. Damaged cells and variable growing conditions can further affect phenotypic expression resulting in inconclusive results. The limitations of phenotypic tests were illustrated in a UK study designed to assess reproducibility between laboratories in the identification of coliform bacteria. The study concluded that phenotypic methods do not provide a reliable method for assigning a species name to test organisms, even for duplicate samples2.
Genotypic methods, by comparison, are not affected by growing conditions or the stress status of organisms. In the same UK study, an automated ribotyping method was able to correctly identify organisms consistently and easily recognised duplicate samples2.
Today, DNA fingerprint identifications can be performed routinely by using sequencing technology or by an automated ribotyping system, such as the DuPont Qualicon RiboPrinter system available from Oxoid in Europe and Australia. The RiboPrinter system offers the advantages of minimal operator intervention, walk-away processing and reliable results in just 8 hours. In addition, unlike alternative methods, the RiboPrinter system is able to characterise organisms to provide valuable strain level identifications.
Sequencing methods use highly conserved genetic information, usually within the rRNA 16S or 23S genes, to provide an accurate, species-level identification. Similarly, the RiboPrinter system uses the highly conserved information within the 16S, 23S and 5S rRNA genes. However, in addition to this, it also uses less conserved intergenic and flanking information. It is in this less conserved information that any variation between strains is found. As a result, the RiboPrinter system is able to detect small differences or similarities between isolates of the same species, allowing characterisation of the organisms and valuable sub-species, or strain-level, identifications.
Identification is made by comparing the isolate's RiboPrint Pattern to the system's extensive database of more than 6,000 known RiboPrint Patterns (containing over 400 new RiboPrint Patterns of critical interest to the pharmaceutical industry) or to the user's customised database.
Pinpointing contamination
When a microbial contaminant is discovered in a pharmaceutical product, a thorough investigation is warranted. This will normally involve analysis of raw materials and the manufacturing environment to try and find the source of contamination. Such investigations may reveal the presence of the same species at several locations. Without further information, it may take many weeks to rectify the problem as each location is addressed. However, the additional sub-species information provided by microbial characterisation is able to match the strain found in the final product to its precise source, allowing faster, more targeted action to eliminate the problem and ensuring that production can be resumed as quickly as possible.
The case studies that follow are real-life examples, however, we have omitted company names for reasons of confidentiality.
Case study 1
A pharmaceutical company faced a potential product recall when an isolate was found to grow during sterilisation method testing. With just two weeks to resolve the problem and over 800 samples to test, the challenge seemed daunting. However, using the Ribo-Printer system, the task was completed easily within the timeframe. It was shown that the problem was not product contamination but an environmental problem within the testing laboratory. Phenotypic testing or 16S rRNA sequencing could not have identified this. The product was proven safe and production was allowed to continue.
Case study 2
A bacterial contaminant was found in an asthma inhaler formulation during the final stages of clinical trials. Phenotypic methods failed to identify the contaminant or to find the source of the problem. With the trial halted, each day that went by increased the delay in bringing the product to market. Turning to the Ribo-Printer system for help, within 8 hours the culprit was identified as Enterobacter cloacae – an organism that had dangerous implications for the target users of the product. Therefore, finding the source became a matter of urgency. Several different strains of E. cloacae were found in several of the raw materials, but only one strain was found in the final product. The source of the problem was found to be the inert carrier for the active ingredient. Once the company switched to a higher grade product, the problem was resolved and trials could be resumed.
In addition to reacting quickly to sterility problems, manufacturers may also use ribotyping and microbial characterisation to monitor the manufacturing environment proactively for potential contaminants. This is a critical cGMP function for both sterile and non-sterile pharmaceutical manufacturers.
Key areas can be monitored regularly for the presence of “high risk” isolates. Each sample analysed by the RiboPrinter system is automatically compared with every previous pattern run on, or added to, that system, allowing users to perform detailed historical tracking. Additional information and comments about each sample, including the location it was isolated from, can be stored with its RiboPrint pattern. Then, if a problem organism reoccurs, operators can find out quickly where and when it appeared before and whether further action is required. Such knowledge allows manufacturers to gain greater understanding and control of the microbial environment within their facilities.
Sharing information
New Data Merging Workstation software further enhances the monitoring capabilities of RiboPrinter systems, combining records from several systems within a firewall into one integrated database to allow even more advanced microbial tracking. For companies with several manufacturing sites around the world, such sharing of information could prove invaluable. For example, if a sterility issue is found to be a problem in every manufacturing site, then a raw material common to each site is likely to be the source of the problem. However, if the problem is confined to one site, the source is more likely to be environmental.
For pharmaceutical and other US Food & Drug Administration-regulated industries, it is essential that any system that stores data is compliant with the Code of Federal Regulations for electronic record security (21 CFR Part 11). The Windows-based RiboPrinter system software meets this requirement with, for example, four levels of security that determine user access to certain features, along with detailed audit trails to track and record any changes to data.
Identification of problem micro-organisms within a day, instead of a week or more; faster pinpointing of the source of contamination; and accurate, informed monitoring of key areas within the production environment all contribute to the reduction or prevention of financial losses, minimising production downtime and protecting the investment of years of research, development and trials.