Knowing your environment includes being aware of critical control points along the processing line, having confidence in quality control procedures and being sure of suppliers. However, even the strictest precautions can be breached and when microbial contamination is discovered in an end product the quest begins to find the source and to eliminate it as quickly as possible.
The development of an automated ribotyping method (The RiboPrinter microbial characterization system, DuPont Qualicon) has taken knowledge of the pharmaceutical environment to a new level. By providing important strain-level information, the system can be used to pinpoint the source of a microbial contaminant with great accuracy and speed. The necessary action can then be taken in that particular area, saving time and resources and allowing manufacturing to recommence without further delay.
Ribotyping provides a fingerprint of the portion of the bacterial genome that codes for rRNA. Conventional ribotyping can take up to 5 days to perform, making it unsuitable for routine testing. Furthermore, since it is a highly subjective method that requires significant manual intervention, variation between laboratories and even between technicians is almost inevitable.
THE PRINCIPLES OF RIBOTYPING
Ribotyping is a genetic fingerprint of the bacterial genomes that code for ribosomal ribonucleic acid (rRNA), providing a highly accurate, strain-level identification.
Restriction enzymes are used to cut the highly conserved rRNA genes and surrounding genetic material into fragments. These fragments are separated by gel electrophoresis, according to their size, and the resulting banding pattern, or fingerprint, is visualised using genetic probes.
The RiboPrinter microbial characterization system, however, is a fully automated ribotyping method that eliminates the need for manual intervention and reduces the time required to perform the ribotyping process to just 8 hours. Any level of microbiologist can run the system easily and there is no need for molecular biology or genetic expertise.
Since the RiboPrinter system has standardised ribotyping through automation, the potential for user variation has been removed and it is now possible to compare samples in time, between operators and even between locations.
THE RIBOPRINTER METHOD
Pure culture samples are added to the RiboPrinter system following heat treatment and the addition of a lysing agent. The system then processes the samples automatically, performing DNA fragmentation, separation of the fragments by electrophoresis, transfer to a membrane, hybridisation of the fragment bands and detection and capture of a digitised image.
Finally, the RiboPrinter performs data analysis, comparing the resulting RiboPrint pattern to the integral DuPont Identification Database (or the user's custom database) to produce a characterisation/identification report. This sophisticated data analysis eliminates the need for subjective interpretation of results, thus enhancing precision and reproducibility.
Unlike some alternative molecular methods the RiboPrinter system can identify micro-organisms to strain level. DNA sequencing reports the sequence of nucleotide base pairs along a length of DNA. In the case of 16S rRNA sequencing, it is the highly conserved 16S gene that is sequenced. As a result, this method provides a highly accurate species-level identification but it is less able to match or differentiate two distinct strains of the same species.
In addition to using the information within the 16S gene, the RiboPrinter also uses other restriction sites along the entire operon. This encompasses the 16S, 23S and 5S genes and all the genetic information in between. It is in this flanking information, which is less highly conserved, where variations between strains will be found. As a result, the RiboPrinter is able to detect small differences or similarities between isolates of the same species, thus providing an accurate strain-level identification fingerprint.
Strain-level information can be used in the pharmaceutical industry to track the source of a microbial contaminant. The RiboPrinter can be used for strain-level tracking, either reactively or proactively.
As a reactive method, the RiboPrinter provides a rapid response mechanism. When there is a sterility failure in the end product, a RiboPrint is obtained of the contaminant. The user then proceeds to test the environment. Although the same species may appear at various points in the processing line, the RiboPrinter identifies the particular strain which exactly matches that found in the end product. This allows the necessary corrective action to be swift and targeted.
Alternatively, the RiboPrinter system can be used in a proactive manner by producing a microbial map of the production plant. To do this, samples are taken at strategic points throughout the facility. This provides a map of all the isolates found in the plant and their exact location. Then, if there is a sterility failure, the problem isolate can be compared to those in the database of known isolates in the plant. The system can then tell them where and when that particular strain was seen before.
Without this information, manufacturing would stop and considerable time and resources would be spent examining the processing environment. Microbial mapping can save a great deal of investigative time and, since manufacturing can resume much more quickly, there are also economic implications.
A major requirement of FDA-regulated industries is that systems used to record data are compliant with 21 CFR Part 11 for electronic record security. The RiboPrinter system satisfies all of these requirements. The system's new Windows-based software includes four levels of security that determine user access to certain features, along with audit trails that track and record changes to data.
Regulations also state that the pharmaceutical industry must validate equipment to show that it is accurate and reliable. An easy to use and comprehensive Validation Package is available for the RiboPrinter system that allows a fully validated method to be implemented in as little as 8 weeks.
The RiboPrinter microbial characterization system has proven its worth in the pharmaceutical industry as a system that allows companies to know more about their processing environments. As more and more of the major pharmaceutical companies are adopting this method in their quality control programmes, it is evident that the RiboPrinter can be put to good use in other areas of the industry, such as anti-infective clinical trial support, research and development, product development and culture integrity.
The full potential of the RiboPrinter system in the pharmaceutical Industry may yet to be realised but when it comes to knowing exactly what is going on in your business, from a microbial point of view, it is beyond compare as a valuable source of information.