Wednesday, February 22, 2012

ECA Hosts the Validation of Molecular Biological Methods Conference

On February 14-15, 2012, the European Compliance Academy (ECA) hosted a two-day conference and exhibition focusing on the validation of molecular biological methods. The Development of Molecular Biological Methods has made a rapid progress during the last several years. Being primarily science based, they found their way more and more to industrial applications. Especially in the food and pharmaceutical industry, Molecular Biological Methods for the detection of adventitious biological agents and impurities gained importance in process control strategies and release testing.

I started the conference with an overview of the future of rapid molecular methods, focusing on nucleic acid amplification and gene sequencing technologies, validation strategies and regulatory expectations. FDA and EMA enablers for method validation and submissions were discussed in great detail, including the recently introduced European Post Approval Change Management Protocol, which is very similar to FDA’s comparability protocol. Applications and specific testing points for bioprocessing and fill-finish operations were discussed. This was followed by an in-depth overview of commercially available technologies and the scientific core principles that make them work.

Emiliano Toso, Merck Serono, followed with a presentation on molecular biology methods for viral safety. He discussed the perceptions associated with certain detection technologies, recent incidents of viral contamination in pharmaceutical facilities (e.g., Mouse Minute Virus; MMV and Vesivirus), and strategies for implementing a viral safety program. Dr. Toso concluded that accurate assay design and vliadtion, trained personnel and GMP compliance could allow molecular methods for viral detection to be a smart and fast solution for monitoring biological processing and reduce the risk of large scale viral contamination.

Next, Christine Farrance, Accugenix, discussed strategies for using multi or single-locus sequence typing (M/SLST) to increase the resolution of microbial identification and strain typing for environmental isolates. Dr. Farrance stated that by combining an accurate method of genotypic identification, such as 16S sequence-based analysis, with M/SLST, it is possible to resolve some of the most difficult organisms to trend and track. This is achieved by analyzing essential outer membrane protein coding genes or housekeeping genes that encode for proteins necessary for the normal cellular functions of bacteria, all of which contain variability in their sequences. M/SLST advantages include the ability to differentiate to the strain or subspecies level even when the organisms are very closely related, as well as being highly reproducible. She included case studies describing strain typing of Pseudomonas, B. cereus and B. subtilis using specific target genes such as gyrB, glpF, pur and pycA. Dr. Farrance concluded that by using M/SLST, the resulting data could be used to support root cause analysis of environmental excursions and even sterility failures.

Anna Gottlieb, National Institute for Biological Standards and Control
Health Protection Agency, provided an overview of quality control reagents for clinical microbiological diagnostic nucleic acid amplification assays. Nucleic acid amplification technologies are now replacing traditional techniques in the clinical microbiology setting, especially when it comes to virology testing. The challenge has been to control the diagnostic kit working reagents. She shared data associated with the variability in organism detection, such as Neisseria, HSV-2, EBV and FLU-B. Simply, there is a need for standardization to reduce both intra-lab and inter-lab variability associated with this type of diagnostic testing. An online QC result reporting system for diagnostic testing was demonstrating, highlighting the ability to monitor and trend laboratory variability while using these methods.

The next speaker was C. Micha Nubling, Paul Ehrlich-Institut, who discussed designing of screening nucleic amplification technologies (NAT) and the implications for variable pathogen targets. NATs are limited in that there is variability between assays because there is a lack of common calibrators. Additionally, false-negative results occur, despite the presence of the target, due to a variety of factors, including limitations in the design of the test as well as capable detection limits. An overview of the efficacy of NAT screening for HIV-1 markers in blood (from donors) within the European Union highlighted the need for more reliable and sensitive methods, including dual-target screening assays.

Stefan Meinzinger, Life Technologies, presented a review of the importance and challenges associated with host cell residual DNA test methods. Regulatory authorities require that residual host cell DNA quantities contained in final dosage forms follow certain guidelines, such as certain log reductions following each purification stage. Method validation strategies and case studies were provided, in addition to an overview of the company’s residual DNA quantitation system.

The next speaker was Melanie Stormer, Paul Ehrlich-Institut, who discussed the challenges in validating molecular biological methods for microbiological control of advanced therapy medicinal products (ATMPs). Microbiological control during the manufacturer of cell-based products is critical, in that exogenous contamination can occur during donation and manufacturer, the source material and final product cannot be sterilized, the shelf-life of the product is extremely short, and the sterility of the source material cannot be guaranteed. Therefore, the development of novel approaches for reliable rapid control testing is vital. Dr. Stormer shared a varety of currently available rapid method technologies for the detection and quantitation of bacteria as well as for Mycoplasma. Guidelines for Mycoplasma testing using PCR, including Ph. Eur. 2.6.7 and 2.6.21, were discussed. Additionally, case studies using EDQM Standards for NAT testing were reviewed.

Holger Kavermann, Roche Diagnostics, provided an overview of a real-time PCR method for the rapid testing for MMV in unprocessed bulks of biotech APIs. ICH Q5A states that in vitro screening assays, using one or several cell lines, are generally employed to test unprocessed bulk, and if appropriate, a PCR test or other suitable method should be used. The EMA guideline on virus safety evaluation of biotech investigational medicinal products states that consideration should be given to the inclusion of a test for MMV if the cell line is permissive for this virus. Additionally, the EMA guideline specifies that the sample to be tested should include cells, when appropriate, and tests should include in vitro and PCR-based screening tests for adventitious agents and an estimation of retroviral particles, where applicable. FDA CBER’s points to consider in the manufacturer and testing of monoclonal antibody products for human use states that bioreactors containing hamster cells can become contaminated with minute virus of mice that may escape detection in routine in vitro assays. Therefore, testing for MMV in unprocessed bulk samples of biotech products is recommended, and specific PCR-based assays are an acceptable alternative to the traditional in vitro assays. Dr. Kavermann described a test design for a MMV PCR assay that included sample preparation, amplification and detection, and control systems, using the company’s system. Case studies in specificity, limit of detection, robustness and comparability with the traditional method were provided.

Next, Thomas Meindl, Labor L+S AG, described the workflow for a MALDI-TOF mass spectrometry sstem for microbial identification. Case studies of tests for accuracy, precision and robustness using clinical isolates and reference strains were discussed. Dr. Meindl stated that MALDI-TOF is a useful tool for microbial identification in GMP-environments, providing fast and accurate results.

Eric Abachin, Sanofi Pasteur, discussed a PCR microarray-based method for the detection of Mycoplasma. He described the science behind the microarray system, which is capable of detecting and identifying 40 different species of Mycoplasma. DNA is extracted from the sample, PCR is performed, and then the amplicons are hybridized onto the microarray. The system then scans the microarray and looks for a positive response. A validation strategy was discussed, which included specificity, robustness and detection limit. Validation data from additional studies using the cell supernatant and crude harvest of vaccine product batches were also provided.

Next, Geert Verdonk, MSD, provided an overview of points to consider when choosing a qPCR method for the detection of Mycoplasma. He described a hybrid approach, using culturing in combination with qPCR. The cell culture method is compatible with several types of samples and the validation with DNA isolation is straightforward. The DNA isolation steps are fast and can be automated. The subsequent detection of Mycoplasma DNA by qPCR offers a low detection limit, is very specific (depending on the technology), and is quantitative or qualitative. He finished with an overview of using statistical methods and a most probable limit (MPL) assay when conducting limit of detection validation studies.

The final speaker was Dirk Vollenbroich, Minerva Biolabs, who presented an overview of validation expectations for Mycoplasma PCR systems. Organism specificity as well as sample matrix specificity test data was discussed, in addition to detection limit and robustness. The presentation ended with a case study on detecting Mycoplasma from a variety of sample matrices, including CHO cells, autologous cell transplants, DMSO and trypsin. The presenter concluded to qualify method sensitivity strictly to 10 CFU/ml, consider validating cell culture enrichment, if possible, focusing on sensitivity but also lab/person reproducibility, and pay attention to the quality of the reference material.

The meeting was very successful, and I encourage you to attend future conferences of this type.

Wednesday, February 15, 2012

Our February Newsletter is Now Available

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New PDA Journal Publication Describes the Validation of a Rapid, Growth-Based Sterility Test

Luis Jimenez and his colleagues at Immunomedics, Inc. have published a very comprehensive review of the validation of a growth-based rapid method as an alternative to the compendial sterility test. The full reference and abstract are provided below.

2012. Jimenez, L.; Rana, N.; Amalraj, J.; Walker, K.; Travers, K. Validation of the BacT/ALERT 3D System for Rapid Sterility Testing of Biopharmaceutical Samples. PDA Journal of Pharmaceutical Science and Technology. 66(1): 38.54.

The BacT/ALERT® 3D system was validated to determine the sterility of different types of biopharmaceutical samples such as water for injection, unprocessed bulk, and finished bulk. The installation, operation, and performance qualification were completed and verified under good manufacturing practices. During the installation and operation validation stages, the functionality and security of the system and software were completed and verified. For the performance qualification, 11 microorganisms were evaluated, six compendial (Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, Candida albicans, Aspergillus niger, Clostridium sporogenes), one representing the number one microbial species in sterile product recalls (Burkholderia cepacia), and four environmental isolates (Kocuria rhizophila, Staphylococcus haemolyticus, Methylobacterium radiotolerans, and Penicillium spp.). Nine of the microorganisms were spiked into three different types of biopharmaceutical samples by three different analysts on different days to ascertain the equivalence, ruggedness, sensitivity, time of detection, and repeatability. In all samples, the BacT/ALERT® exhibited equivalent or better detection than the standard test. With the exception of M. radiotolerans, all 11 microorganisms were detected within 2.5 days using the BacT/ALERT® system and the standard test. The detection times for M. radiotolerans in the three sample types averaged 5.77 days. The minimum detectable level of cells for all the microorganisms tested was found to be within 1 to 2 CFU. The system optimized sterility testing by the simultaneous on-line, non-destructive incubation and detection of microbial growth.

Wednesday, February 1, 2012

Standardization Key to Taking Next-Generation Sequencing (NGS) to the Next Level

Genetic Engineering & Biotechnology News ( believes that as NGS technology makes steady progress with faster and cheaper machines, the regulations will have to keep up with this pace.

As next-generation sequencing (NGS) moves into medicine, its widespread adoption means that sequencing machine manufacturers will require FDA approval to use these instruments in the clinical setting, and test manufacturers will need to adapt approved diagnostics to these platforms. Certain performance specifications will thus need to be established and used to ensure reliable test results.

Created in 2010 at the Center for Disease Control, the Laboratory Science, Policy and Practice Program Office (LSPPPO) is intended to “provide leadership, advocacy, and cross-cutting services to continuously strengthen the quality of laboratory science, policy, and practice at CDC, in the United States, and globally.” Part of this mission includes assistance to laboratories when implementing next-gen sequencing into their practice settings.

To facilitate the effective and appropriate use of next-gen technology, CDC convened a national workgroup for the Next-Generation Sequencing Standardization of Clinical Testing (Nex-StoCT) in April, 2011. Its Genetics Team along with the newly-established Nex-StoCT working group is developing a manuscript that, the organization says, will present “evidence- and quality management-based recommendations for attaining an analytically valid test result and meeting regulatory and professional practice standards for NGS.”

In 2010, the EuroGentest Validation Group published its paper outlining the principles of validation and verification in the context of human molecular genetic testing. The paper describes implementation processes, types of tests and their key validation components, and suggests relevant statistical approaches that can be used by individual laboratories to ensure that tests are conducted to defined standards.

As the CDC and the EuroGentest Validation Group continue to develop standards for the new world of gene-based clinical testing, the two major contenders for the clinical market, Life Technologies’ Ion Torrent and Illumina’s MiSeq, are focusing on FDA approval for their devices and the delivery of validated clinical tests adapted to their sequencing platforms.

Commercializing PGM Sequencer

Life Technologies’ Andy Felton told GEN that the company will first see CE-IVD clearance for the Ion PGM™ Sequencer in Europe in 2012 and then will pursue FDA clearance in the U.S. “We believe there is a lot of demand and we wouldn’t take this step unless we thought the market was ready for next-gen sequencing technology,” he said. “We have had a year on the market to stabilize the platform and to make improvements. The platform is ready to be taken forward.”

Since the PGM sequencer was launched in 2010, with a 10 Mb output on the Ion 314 Chip, a 127 Mb dataset has been released on the same chip. This shows, according to the company, greater than 10x scaling over nine months. The company has also released a 1 Gb dataset on the new Ion 318 chip, demonstrating 100x scaling of the platform in under a year.

Felton also said that “there has been incredible interest among players in diagnostics, a ground swell of demand in large and small companies for a regulated version of a next-gen sequencer. What this means for some of these companies is that they would develop a test and put it onto approved next-gen platforms.”

In October, Life Tech announced availability of its new Ion AmpliSeq™ Cancer Panel, the first product utilizing Ion AmpliSeq technology. It covers oncogenes and tumor suppressor genes. Standardization, Felton said, will boost individual technologies because the reagent and test have been through rigorous tests, providing confidence in answers given by a particular platform. “We believe there will be a large number of tests going forward, and we think the Ion PGM Sequencer is particularly suited because of its flexibility and because and you can scale the experiment to the chip.”

Felton expects applications for Ion PGM Sequencers will grow rapidly in areas like somatic mutation detection for cancers, resequencing for inherited diseases, and mendelian disease research. “Microbial applications will likely come a little bit later except in epidemiological use,” Felton added.

Validating TruGene on MiSeq

On November 2, 2011, Siemens Healthcare Diagnostics and Illumina announced that they entered into a partnership aimed at setting new standards in the use of next-generation sequencing for the rapid, accurate identification of patients’ infectious disease states and potential treatment paths.

Siemens and Illumina will initially work on making Siemens’ TruGene® HIV-1 Genotyping Assay, one of the first FDA-approved HIV-1 DNA sequencing-based tests, compatible with Illumina’s MiSeq analyzer.

“We had an FDA-cleared product on the market, TruGene, based on a slab gel sequencing technology, for eight years,” said Trevor Hawkins, Ph.D., head of next-generation diagnostics for Siemens’ Healthcare Diagnostics. “We looked at it and said this is a great starting point for Siemens to begin to understand how these next-generation sequencing devices could be used in real clinical settings. We have a large global TruGene community developing a lot of datasets and understanding of how to use sequencing in the clinic.”

He further explained that Siemens “got into this as we wanted to see what it would take to understand how to standardize methodologies, workflow, and IT/automation and answer all the questions on the table, including those related to chemistries and how data management would be handled.

“The key things we have been doing over the last couple of months include validation of results run on a standard TruGene platform versus those run with the MiSeq system and analyzing any differences in the data.” There is so much data that one of the key questions Dr. Hawkins seeks to answer is at what depth and sensitivity levels do you start to use the data in a clinical setting.

Dr. Hawkins also noted that Siemens has “been looking at cloud-based IT solutions; all these devices are cloud-enabled, so what will the impact be of cloud-based systems in the next 10 years? How will the TruGene community use cloud-based tools?”

Siemens is working on analyzing HIV sequences from the traditional platform for its HIV tests and analyzing output for known drug resistance. “The way in which next-gen systems work is inherently different from Sanger-based sequencing,” Dr. Hawkins pointed out. “The issues at hand are ones of using new chemistries and then validating that you are getting the same quality of results over large datasets.

“That is the work that has to go on: sequencing the same sample multiple times using the old technology and the new technology to ensure you are arriving at the same end-point answer at the same or better qualities with the new approaches.”

Siemens plans to go to the FDA this year with its next-gen sequencer-based tests. “Our programs in HIV are going as expected, and we are now looking at what potential assays could be our next challenge in taking next-gen sequencing to the clinic,” Dr. Hawkins stated. He anticipates that the FDA will be a partner in understanding how the devices will be used.

“Next-gen sequencers will change the way in which we interact with healthcare,” he asserted. “Everything we have seen before is going to change over the next five years because of these devices. We formed the partnership because Siemens wanted to be at the table talking to the customers, the community, and the FDA to see a real-world example of a widely used test.”

Investigators studying the use of next-gen sequencing in identifying patients, for example, at risk for breast cancer say that the cost savings in applying such technologies will allow the application of genetic testing to a wider range of individuals than is the current standard.

They also say as more next-generation sequencing technologies become available for genetic testing, results on sensitivity and specificity should be made freely accessible to those who order the test. Hopefully, they say, comparisons of various technologies will also become available.

More Use of Whole Gene Sequencing Poised to Play Important New Roles in Microbiology and Medical Laboratory Testing

DARK Daily Laboratory and Pathology News ( recently predicted that gene sequencing is on a fast track to move from research settings into clinical laboratories, as rapid gene sequencing and whole genome sequencing technologies are giving microbiologists new tools to quickly identify microbes and other infectious disease agents.

In clinical laboratories across the nation, microbiology has greatly benefited from the introduction of molecular diagnostics in clinical practice. Now the field of microbiology is poised to undergo a more profound transformation of clinical practice, due to advances in whole genome sequencing.

Leaders in this field are calling these developments “transformative” and say they have the potential to change “all aspects of microbiology.” The driver to this emerging trend is advanced technology that makes it possible to sequence the whole gene sequence of an organism in a day or less, for a cost that is $1,000 and falling rapidly.

In the past six months, microbiologists and pathologists at such hospitals as Methodist Hospital in Houston, Texas, have begun to do whole genome sequencing of microbes found in specimens collected from patients arriving in the emergency room. The New York Times wrote about these developments in a story titled “The New Generation of Microbe Hunters,” that it published on August 29, 2011.

Medical Laboratories May Soon Be Gene Sequencing Microbes

Medical laboratory managers may be surprised to learn how swiftly this specialized area of microbiology is advancing. In a paper published in the New England Journal of Medicine (NEJM) last summer, David A. Relman, M.D., Professor, Medicine-Infectious Diseases, Microbiology, Immunology, provided information about progress in whole genome sequencing of infectious disease agents.

He provided a list of the current state of microbial genomics. Some of that data follows:

- 1,554 complete bacterial genome sequences (representing mostly pathogens) have been published, with 4,800 more in progress.
- 112 complete archaeal genome sequences have been determined and 90 more are in progress.
- 41 complete eukaryotic genome sequences have been determined (19 from fungi), plus another 1,100 are in progress.
- 2,765 viral species have complete reference genome sequences available.

Relman had several predictions which can be used by clinical laboratory administrators and pathologists to anticipate developments in the field of clinical microbiology. In the NEJM story, Relman wrote that, “Genome sequencing of a microbe or virus will soon be easier than characterization of its growth-based behavior in the laboratory. In the next three to five years, direct shotgun sequencing of the DNA and RNA in a clinical sample may become a routine matter.”

Relman went on to write that, “The power of full-genome sequencing to discriminate between closely related strains and track real-time evolution of disease-associated clonal isolates offers the possibility of tracing person-to-person transmission and identifying point sources of outbreaks.”

This is a role that clinical laboratories can perform. In fact, Relman gave an example of this type of clinical diagnostic application. He stated that, “Using this approach, investigators established a previously unrecognized link among five patients with the same clonal strain of methicillin-resistant Staphylococcus aureus from a hospital in Thailand.”

Many pathologists recall recent events in Haiti. After a half century without a case of cholera in Haiti, in the months following the 2010 earthquake, there was a major cholera outbreak. Gene sequencing of the cholera strain was used to determine that the source of the outbreak was nearly identical to strains circulating in South Asia. Eventually, that cholera outbreak was traced back to United Nations peacekeepers from Nepal. Contamination by fecal matter from their camp infected local water sources. Since then, approximately 520,000 cholera cases have been reported in Haiti, along with about 7,000 deaths attributed to the disease.

Cheaper, Faster, More Accurate Gene Sequencing Can Change Microbiology

These are examples that show how cheaper, faster, and more accurate gene sequencing technologies are poised to transform clinical microbiology. That’s the opinion of Matthew K. Waldor, M.D., Ph.D., of Harvard Medical School. In The New York Times story, he stated that this new technology “is changing all aspects of microbiology. It’s just transformative.”

It was Waldor, in a collaboration with James M. Musser, M.D., Ph.D., Chairman of Pathology and Genomic Medicine at the Methodist Hospital System, who used rapid gene sequencing to investigate the source of the cholera outbreak in Haiti.

Another one of Drs. Waldor and Musser’s contemporaries is Eric Schadt, Ph.D., Chairman of Genetics at Mount Sinai School of Medicine in New York City, and the Chief Scientific Officer at Pacific Biosciences. Schadt is engrossed with making disease “weather maps.” Developed from swabs and samples from public venues such as surfaces in subways and hospitals, as well as from fecal samples in sewage treatment plants, disease weather maps would provide real-time tracking of what bacteria and viruses are present in a particular area and how prevalent they are.

Through genetic testing of sewage, for example, Schadt was able to determine what disease-causing microbes were present from a particular population. “This is like public health epidemiology,” Schadt told The New York Times. “We could start assessing the dietary composition of a region and correlate it with health.”

Collectively, the examples provided in this Dark Daily e-briefing show how quickly microbiologists, infectious disease doctors, and pathologists are applying rapid gene sequencing and whole gene sequencing to a wide range of research and clinical purposes. Pathologists and clinical laboratory executives will recognize that, at this stage in the technology development of these tools, it is not a matter of when, but how fast they will find a place in microbiology and clinical laboratory testing.