Happy 200th Birthday, Robert Bunsen!

Today is Robert Bunsen's 200th birthday. Even Google has developed an interactive doodle that allows you to change the height of the bunsen burner flame depending on where you place your mouse on the search engine's main page! To celebrate, here are the 10 things you need to know about this German chemist:

1. Robert Bunsen was one of the most admired scientists of his generation. He lived to the age of 88.

2. The German chemist developed the bunsen burner in 1855.

3. He never took out a patent on the burner, on a point of principle.

4. Bunsen once almost died of arsenic poisoning during his research.

5. He lost an eye in a laboratory explosion (not sure if his burner was involved!).

6. In 1860 he discovered a new element 'caesium'and a year later he found 'rubidium'.

7. He loved teaching.

8. He retired at the age of 78.

9. Bunsen never married.

10. His grave is in Heidelberg, Germany.

For a more in-depth review of Robert Bunsen and his invention, please visit Tim Sandle's blog at http://pharmig.blogspot.com/2011/03/history-of-microbiology-bunsen-burner.html.

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Raman spectroscopy for rapid identification and enumeration

Andrew Bartko, Battelle Memorial Institute, discussed the use of a novel Raman spectroscopy technology for the rapid identification of microbial contaminants in clean room environments. He provided an overview of how EM samples can be introduced into the Raman system, where each particle or organism cell can be detected, enumerated and identified. Low level detection studies were also discussed, where it was demonstrated that Raman can count and identify multiple types of microorganisms, such as E. coli and C. albicans, in the same sample. The technology is non-destructive, allowing for additional confirmatory analysis if so desired, and can also be adapted to perform microbial identifications on cells that have been stained with a viability substrate, which is what we would normally use with flow cytometry and solid phase cytometry RMMs

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New Changes to EMA's Review of RMM Validation Protocols!

Riccardo Luigetti, EMA, provided an overview of the EMA’s current perspectives on RMMs. Ph. Eur. chapter 5.1.6, Alternative methods for control of microbiological quality, provides the regulatory basis for the introduction of RMMs in the EU. Not only do the various EU competent authorities support RMMs, but some of the elements of the new variations regulations (implemented in January 2010) can be used to support the introduction of RMMs.

Historically, RMM validation protocols and their associated data were submitted under a Type Variation and the competent authorities reviewed all of this information as a whole.

A very recent change to the management of RMM reviews has just been introduced, which should make the validation and approval process much more predictable. The new process, which is very similar to FDA’s comparability protocol, is called the Post Approval Change Management Protocol (PACMP). In this two-step process, a testing protocol is first submitted as a Type 2 Variation, and when the protocol is approved, you perform the testing as specified in the protocol. The protocol should include the overall testing strategy, such as the planned studies, acceptance criteria and methods.

The second step of the PACMP process involves submitting the resulting data (assuming they have met the protocol’s acceptance criteria) as either a Type 1A or 1B Variation. The decision as to whether the data is submitted as a Type 1A versus a Type 1B variation is determined at the time of protocol review and approval. Because this is a new process, the EMA cannot provide examples of data submission requirements at this time; however, if the data is submitted under a Type 1A Variation, you can immediately implement the rapid method, while a Type 1B Variation requires a 30-day waiting period while the data is reviewed. In either case, as long as the data meets the protocol’s acceptance criteria, there should be no surprises during the data review once submitted.

Prior to submitting the PACMP, you can also discuss your strategies with the EMA under the Scientific Advice (SA) Procedure. This procedure was introduced in 2010 and provides an opportunity for a company to request feedback on a proposed validation and implementation plan. The procedure is short in duration (30 days), and the requesting company can be invited for a discussion meeting. The EMA’s response is in the form of an official recommendation letter, although the letter is not legally binding. Following receipt of the SA letter, the company may ask for follow up discussions.

The introduction of the PACMP is a welcomed addition to the EMA’s other recent policy changes, and will greatly simplify the validation and implementation of RMMs within the EU.

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RMMs for Environmental Monitoring

David Jones, Rapid Micro Biosystems, provided an overview of currently available RMM technologies that can be used for environmental monitoring. These included real-time active air monitoring systems based on optical spectroscopy, flow cytometry, solid phase cytometry, ATP bioluminescence, and direct autofluorescence of actively growing microbial colonies.

They also evaluated the efficiency of microbial recovery in the Growth Direct system during active air sampling and surface sampling. They captured samples on membranes that were subsequently transferred to Growth Direct agar cassettes, and the recovered counts were compared with conventional air sampling methods and Rodac plates. The recovery in both the RMM and the conventional methods were comparable.

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Rapid endotoxin testing

Geert Verdonk, Merck/Schering Plough, presented his validation studies using the Charles River Laboratories Endosafe PTS. Dr. Verdonk explored the use of this rapid and portable endotoxin detection system as part of Merck’s PAT-RMM program in an API production environment. Parallel testing on endotoxin spiked samples was conducted against the gel-clot and turbidometric endotoxin procedures. Robustness testing was also evaluated using different operators, sample temperatures, sample volumes, and after “rough treatment” of the system (i.e., after touching the test cartridges, reservoirs and optical cells). The system is now fully implemented and has recently been used to support a root cause investigation in contaminated use points from a purified water system.

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MALDI-TOF Mass Spectrometry and Equivalency Testing

Rosa Siciliano, CNR, provided an overview of MALDI-TOF mass spectrometry. Microbial identification is based on spectral fingerprints between different microorganisms, where some peaks (molecular masses) are specific to Genus, species, and in some cases, subspecies characterization. Additionally, spectra are reproducible as long as the bacteria are grown under the same conditions. Mass spectra are derived from ribosomal proteins, although spectra of surface macromolecules can also be obtained. The strengths of using MALDI-TOF mass spectrometry include the identification of bacterial, spores, yeast and mold, automated analysis, and no need for preliminary information prior to evaluation (e.g., Gram stain result). Some weaknesses include the potential need to grow bacteria to levels that can be detected by the system, operators are required to have a basic level of expertise in mass spectrometry, and it is not possible to obtain multiple identifications from mixed cultures (i.e., a pure culture is currently required).

Mark Blanchard and Deborah Smith, both from Merck Millipore, provided insights on how to demonstrate equivalence between an existing method and an alternative method. They presented three examples of how to demonstrate equivalence: (a) accuracy of matched outcomes, (b) comparing two independent assays near the limit of detection, and (c) evaluating the false negative rate of a presence/absence test. The speakers also discussed the limitations when using low levels of microorganisms (e.g., less than 5 cfu) when demonstrating equivalence between two methods: many of the counts may actual be negative (i.e., 0 cfu), the percent variation is high, and you will need many replicates to accurately estimate the average cell count. The discussions will form the basis of a workshop on this topic tomorrow afternoon. Stay tuned for additional entries!

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Viral Safety and Mycoplasma Testing

Emiliano Toso, Merck-Serono, provided an overview of recent viral contamination events within the industry (e.g., 2009 Vesivirus contamination at Genzyme, resulting in >$300MM in lost sales), and the advantages of using nucleic amplification and PCR-based techniques to rapidly detect the presence of viruses. He explained that the diversity of viruses makes viral detection and their selective destruction difficult. However, the use of rapid viral methods (he calls these RVMs) could provide fast in-process control, thereby reducing the risk of large-scale contamination events with bioprocessing facilities. An example work flow for in-process monitoring of viral contamination includes clarification or concentration of unprocessed bulk harvest material, followed by DNA/RNA isolation and then reverse transcriptase PCR. Using this strategy reduces a 3-month in-vivo viral detection assay down to a 1-2 day qPCR in vitro assay.

Next, Miguel Nogueras, Abbott, discussed the use of rapid methods for Mycoplasma detection. He described the current 28-day assay and how faster testing is required for products with short half-lives (e.g., autologous and tissue based products), raw materials, release testing, process development testing, cell line or virus sock qualification and contamination monitoring. A variety of RMM techniques were described, including nucleic acid amplification, immunological tests, enzyme based methods, and hybridization methods without the need for nucleic acid amplification. He then described Abbott’s strategy for validating a RMM using the following parameters: interference, specificity, detection limit, precision, reproducibility, robustness, system suitability and comparability. The latter established a relationship between the RMM and existing method unit of measurement (e.g., genomic copies, protein or enzyme vs. CFU). Finally, Miguel discussed their strategy for validation and implementation, which included design planning, input, output, verification, validation and transfer. During these phases they were actively engaging the regulatory authorities.

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Start of the 2011 PDA Europe Pharmaceutical Microbiology Conference

This year’s meeting started with a discussion on the role of the European Pharmacopoeia in the use of modern rapid microbiological methods.

Emmanuelle Charton, EDQM, provided an overview of the history and objectives of Chapter 5.1.6, as well as other chapters within the Ph. Eur. that encourages the use of rapid or alternarive microbiological methods. For example, Ph. Eur. 2.6.12 and 2.6.13 states that alternative microbiological procedures, including automated methods, may be used, provided that their equivalence to the Pharmacopoeia method has been demonstrated. Chapter 2.6.7 states that nucleic acid amplification techniques (NAAT) may be used as an alternative to one or both of the other Mycoplasma methods after suitable validation. Chapter 2.6.27, Microbiological control of cellular products, also allows the procedure to be carried out manually or using an automated system.

Next, Hans van Doorne, University of Groningen, provided preliminary results from a questionnaire that was sent to the industry and to European Licensing Authorities (via National Pharmaceutical Authorities; NPA) with respect to the existing Chapter 5.1.6. The questionnaire asked if the current chapter is appropriate and whether a revision is necessary. Because the enquiry is ongoing, the MMM working party has not finalized any decisions, and the Ph. Eur. Commission has not discussed the results, only preliminary information was provided during this presentation.

Question: Which compendial and non-batch release methods have been replaced by alternative/rapid methods?

Response: RMMs are currently used in place of the compendial tests for sterility (2.6.1), microbial enumeration (2.6.12), specified microorganisms (2.6.13), Mycoplasma (2.6.7) and the standardization of suspensions. RMMs are also used for purposes other than batch release, including in-process control, real time release of pre-filtration bioburden, environmental monitoring, trouble shooting, microbial identification, antimicrobial assays and WFI testing.

Question: What do you consider as the strengths of the chapter?

Response: In general, the chapter applies to any new method not described in the pharmacopoeia, it is easily understandable and applicable, and it provides a road map for approval of rapid methods in the EU.

Question: Do you consider the validation example useful

Response: Both the NPA and industry considered generally view the example useful; however, it is difficult to understand and should be more aligned with the rest of the chapter. Additionally, because the section is confusing and conflicts with the previous sections, USP 1223 and PDA TR#33, it can be removed without loss of clarity.

Question: Would you favour having more examples?

Response: Most respondents stated yes. It would be helpful to provide validation examples for the main RMM technologies, and should be aligned with what is normally described in an FDA comparability protocol. Examples could also be provided where the results obtained by the new method differs significantly from the compendial method. Examples of sterility test validation was also requested, in addition to more examples on the use of statistical methods under a variety of conditions.

Question: What do you consider the weaknesses of the chapter?

Response: There is no global harmonization with other published documents (USP 1223 and PDA TR#33). It is also too prescriptive, and microbial identification methods should be separated from true rapid methods. There were also questions raised about the equivalence between the chapter and EN ISO methods.

The presentation concluded with some rationales for a revision. There is a need to address the use of alternative methods with Process Analytical Technology (PAT) concepts. Next, there needs to be a clearer distinction between methods for isolation and detection vs. methods for identification. Furthermore, there should be more distinction on the discriminative power of identification methods (Genus vs. species), an improvement and expansion of the example section, and terminology should be harmonized with international standards.

I rounded out the first session with a discussion of industry perceptions that have prevented or delayed many companies from validating and implementing RMMs. Some of these perceptions include (a) little or no regulatory guidance, acceptance or understanding of RMMs, (b) we will see things we have never seen before and our products will be at risk, (c) we will have to change our acceptance levels or specifications, (d) there is no clear guidance on validation expectations, and (e) we will realize little or no return on investment. Each of these perceptions were thoroughly discussed and dismissed, and I provided guidance on how the current worldwide regulatory framework actually encourages the qualification and implementation of RMMs.

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Blogging from the PDA Europe Microbiology Conference

Starting tomorrow, I will be blogging from the 2011 PDA Europe Microbiology Conference in Berlin, Germany. The conference lasts for two days and will feature a number of rapid micro method presentations. Featured speakers includeans van Doorne (Univ. Groningen), Emmanuelle Charton (EDQM), Klaus Haberer (Compliance GmbH), John Metcalfe (US FDA), Riccardo Luigetti (EMA) and Barbara Potts (Biologics Consulting Group). I will also be delivering my perspectives on the future of microbiology testing and the role of rapid methods on pharmaceutical manufacturing and the quality of medicinal products.

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Miniaturized isothermal nucleic acid amplification, a review

A recent article in the publication Lab Chip provides a review of miniaturized analysis systems using alternatives to PCR. The abstract from authors Asiello and Baeumner is as follows:

Micro-Total Analysis Systems (µTAS) for use in on-site rapid detection of DNA or RNA are increasingly being developed. Here, amplification of the target sequence is key to increasing sensitivity, enabling single-cell and few-copy nucleic acid detection. The several advantages to miniaturizing amplification reactions and coupling them with sample preparation and detection on the same chip are well known and include fewer manual steps, preventing contamination, and significantly reducing the volume of expensive reagents. To-date, the majority of miniaturized systems for nucleic acid analysis have used the polymerase chain reaction (PCR) for amplification and those systems are covered in previous reviews. This review provides a thorough overview of miniaturized analysis systems using alternatives to PCR, specifically isothermal amplification reactions. With no need for thermal cycling, isothermal microsystems can be designed to be simple and low-energy consuming and therefore may outperform PCR in portable, battery-operated detection systems in the future. The main isothermal methods as miniaturized systems reviewed here include nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), and strand displacement amplification (SDA). Also, important design criteria for the miniaturized devices are discussed. Finally, the potential of miniaturization of some new isothermal methods such as the exponential amplification reaction (EXPAR), isothermal and chimeric primer-initiated amplification of nucleic acids (ICANs), signal-mediated amplification of RNA technology (SMART) and others is presented.

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