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DNA present and an amplicon melting temperature (Tm) in a specific range. The assay also includes a Discriminatory Positive/Extraction Control, which is a large plasmid containing a Mycoplasma DNA sequence. The Tm of the Discriminatory Positive/Extraction Control is different than the Tm of Mycoplasma DNA, eliminating the possibility of a false positive test result due to accidental cross-contamination of a test sample with control DNA. The assay uses a negative control, positive control, extraction control, and inhibition controls to ensure the validity of test results.
The Real-Time PCR Mycoplasma testing procedure complies with PhEur 2.6.7 Mycoplasmas, which specifies requirements for validation of a nucleic acid-based test [4]. The validation study tested drug product samples derived from multiple chondrocyte strains spiked with viable organisms (and corresponding DNA solutions) from six Mycoplasma species. This method is a qualitative test for the presence or absence of microorganisms, similar to a sterility test for bacteria and fungi. Specificity and Detection Limit are the most critical validation parameters for this type of qualitative test. USP <1223> Validation of Alternative Microbiological Methods and PhEur 5.1.6 Alternative Methods for Control of Microbiological Quality specify additional validation parameters for a qualitative test including Robustness, Repeatability, and Ruggedness [5, 6] (ICH has not yet addressed alternative microbiological methods). PhEur 2.6.7 also requires an assessment of Equivalence, since the Real-Time PCR method is a replacement for the
conventional culture method [4]. Table 1 contains a summary of the validation study design.
Lessons Learned – Validation
Overall, the validation experiments went smoothly given the complexity of the study and the logistics of execution. Even so, it is important to look for opportunities for continual improvement when working in a cGMP production environment. This validation presented three such opportunities in the areas of reference standards, logistics, and detection criteria.
Reference Standards
Mycoplasma Reference Standards
Now Available
• Genomic Mycoplasma DNA Preparations (Cat. No. M-1250)
• Non-viable Mycoplasma Lysate Preparations (Cat. No. M-1200)
• Viable Mycoplasma Reference Strains (Cat. No. M-1100)
Bionique.com 518-891-2356 For Research Purposes Only – Not for diagnostic and clinical use
98 | | September/October 2013 - 15TH ANNIVERSARY ISSUE
Supporting your rapid mycoplasma detection method with multiple convenient formats and unsurpassed customer support
Preparing reference standards containing a well-characterized quantity of correctly-identified viable Mycoplasma organisms requires specialized techniques [7]. Commercial sources of Mycoplasma organism reference standards characterized in terms of their genomic DNA copy to colony forming unit (GC/CFU) ratio [8] are currently available from multiple suppliers. Determining the GC/CFU ratio is necessary to establish whether an alternative nucleic acid test yields results equivalent or better than a compendial method. It is important to reduce the nucleic acid contribution from non-viable organisms because DNA detection is not necessarily equivalent to viable organism detection. Culture methods detect only viable organisms, while the Real-Time PCR test detects Mycoplasma DNA from both viable and non-viable sources. With knowledge of the GC/CFU ratio, it is possible to establish the detection limit solely using purified Mycoplasma DNA reference standards, which are also now commercially available. Neither Mycoplasma organism reference standards characterized in terms of GC/ CFU nor purified DNA was available for the validation study conducted in 2011.
Overcoming this hurdle required working closely with a contract laboratory specializing in growth and detection of Mycoplasma. The laboratory used stock solutions from organisms harvested during log-phase growth to maximize viability. They prepared viable Mycoplasma organism solutions at a titer of approximately 1000 CFU/mL from these stock solutions and confirmed the actual titer by plate count. They also prepared non-viable lysed organism solutions (unpurified DNA) from the viable organism solutions equivalent to 1000 CFU/mL. The validation report calculated the GC/CFU ratio from calibration curves correlating number of genome copies (GC) and threshold cycle (Ct) based on an average
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