By: Yoann Mainguy, Global Product Manager, Sterility & Mycoplasma Testing, and Yoann Gatineau, Technical Leader, Mycoplasma Testing; Millipore SAS, Molsheim, France (an affiliate of Merck KGaA, Darmstadt, Germany)
On 1 April this year, the Ph. Eur. Chapter 2.6.7, in its thoroughly revised form, came into force. It applies to medicinal products derived from or manufactured using living cells or biological systems and that have a realistic risk of contamination by Mollicutes (including the genus Mycoplasma).
One of the most important changes is that the new Ph. Eur. 2.6.7 explicitly recognizes nucleic acid amplification techniques (NAT) such as PCR as standalone methods after validation and proof of suitability for the specific product. Ph. Eur. 2.6.7 now goes beyond current USP regulations, where chapter <63> still considers NAT as alternative methods but where the draft general chapter <77> on NAT-based testing suggests that changes along similar lines will come. NAT methods carry the significant advantage of much earlier results than traditional culture-based mycoplasma testing methods, which usually take several weeks.
How CFU (Colony-Forming Units) and GC (Genome Copies) relate
One of the challenges is to demonstrate the limit of detection of the method, which Ph. Eur. 2.6.7 stipulates must not be higher than 10 CFU/mL for any product matrix being tested. Because NAT do not generate results in CFU, Ph. Eur. 2.6.7 has introduced genome copies (GC) as a unit alongside CFU. Although they correlate, these two measures differ in what they cover. Beyond viable cells, the GC value can also include dead or damaged cells, or even free DNA fragments. CFU, on the other hand, takes no account of non-culturable mycoplasmas and counts several cells as only a single CFU if they clump or aggregate. For comparability, the difference between GC and CFU should be as low as possible, which is why the new Ph. Eur. 2.6.7 stipulates that reference standards serving as controls and references must be based on mycoplasma cells in the exponential (or mid-log) growth phase, when divergence tends to be lowest. Accepting the reality that GC will nevertheless regularly be higher than CFU, the Ph. Eur. 2.6.7 has set a pragmatic upper limit of 10 for the GC/CFU ratio of reference preparations for NAT-based methods, which is mandatory unless otherwise justified. As a consequence, the limit of detection for NAT-based methods must regularly be below 100 GC/mL. This allows for good comparability across different testing methods and laboratories, thereby enhancing the reliability of NAT results.
Requirements for mycoplasma reference standards
Preparing a mycoplasma reference standard inoculum that fulfills the regulatory requirements is, however, a challenge that requires expertise in mycoplasma expansion, harvest and nucleic acid titration. Such standards have to meet a number of requirements, including the confirmation of the strain’s identity at the species level, with DNA sequencing highly recommended. They must be traceable to reference cultures of mycoplasma reference strains and not be more than 15 passages away from the original strain, so data about the subculture history should be available. The titer in CFU, as well as the number of GC must be determined prior to freezing the working suspensions or dilutions. These numerous requirements have led many QC labs to turn to commercially available mycoplasma standard strains, which offer the added advantages of convenience, time savings and a lower contamination risk because the handling of these bacteria is minimized.
Mycoplasma reference standards can be used as positive controls for various activities in method validation and routine use of all methods described in Ph. Eur. 2.6.7, USP <63> or JP <G3-14-170> (see tables), including in-process and release testing. The readily calibrated Mycosafe® Mycoplasma Reference Standard strains we distribute, come as either viable cells or, for uses where handling cultivable mycoplasmas is not required or not permitted, heat-inactivated cells that are intact but not cultivable. All these deep-frozen and CFU-calibrated preparations have a defined GC/CFU ratio of below 10, making them ready for immediate use once thawed.
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Which format should be used in method validation?
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Activity
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Description
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Recommended format
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Generic validation
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Demonstrates that the method can consistently detect mycoplasmas according to regulatory requirements. Can be used and extended to a broad range of matrices, as long as the main process, including instrumentation, remains unchanged.
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Viable cells recommended in case of side-by-side comparability study
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Comparability study
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Demonstrates equivalence of the method with compendial culture-based methods using a performance comparison.
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Viable cells required
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Test for inhibitory substances
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Product-specific test for inhibitory substances, to be performed with a reduced number of mycoplasma reference standards.
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Viable cells not required
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Which format should be used in routine use?
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Activity
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Description
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Recommended format
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Cell-based enrichment followed by NAT
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The test sample and a suitable cell substrate (as described under the indicator cell-culture method) are cultured together for a suitable period. The nucleic acids are then extracted from the cells and used for detection by NAT.
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Viable cells required
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Media-based enrichment followed by NAT
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The test sample is inoculated into liquid media suitable of supporting growth of the pharmacopoeial species and incubated for a suitable period. The nucleic acids are then extracted from the enrichment culture and used for detection by NAT.
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Viable cells required
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Direct NAT
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The nucleic acids are extracted from the test sample and used directly for detection by NAT (without cell- or media-based enrichment).
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Viable cells not required but DNA standard not recommended
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