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.