by Dr. Ray Nims
The European Pharmacopoeia chapter 2.6.7 Mycoplasmas, begining with version 5.8, has provided a mechanism for replacement of the current 28-day culture method for detection of mollicute (mycoplasma and acholeplasma) contaminants in biopharmaceutical bulk harvest samples with more rapid, nucleic acid-based, methods. The US FDA has yet to provide formal guidance on this topic, although it has become clear that the agency is willing to consider such methods, provided that they are shown to be equivalent to or superior to the current approved methods.
The European Pharmacopoeia chapter 2.6.7 Mycoplasmas, begining with version 5.8, has provided a mechanism for replacement of the current 28-day culture method for detection of mollicute (mycoplasma and acholeplasma) contaminants in biopharmaceutical bulk harvest samples with more rapid, nucleic acid-based, methods. The US FDA has yet to provide formal guidance on this topic, although it has become clear that the agency is willing to consider such methods, provided that they are shown to be equivalent to or superior to the current approved methods.
For biopharmaceuticals, a satisfactory outcome in a mycoplasma detection assay which is compliant with European Pharmacopoeia 2.6.7 or the 1993 FDA Points to Consider guidance is required on a lot-by-lot basis. Of the various lot-release assays performed on each given lot of a biopharmaceutical, this particular test is typically the most lengthy. Expediting the lot-release process through replacement of the 28-day approved culture test with a rapid mycoplasma detection test is therefore a strong motivating factor for the biopharmaceutical industry.
Figure. The MicroSEQ Mycoplasma assay provides a level of detection less
than 10 CFU/ML.
In addition, several vendors are now offering mycoplasma detection kits which will allow biopharmaceutical entities to perform rapid mycoplasma testing in-house. For example, Life Technologies offers the MicroSEQ® Mycoplasma Detection Assay, Roche Applied Science offers the MycoTool™ PCR test and Millipore offers the MilliPROBE® mycoplasma detection system.
It is incumbent upon the biopharmaceutical company to demonstrate comparability between the rapid mycoplasma method and the current approved culture method for each product matrix for which a rapid method is proposed. Guidance on such comparability testing is provided in the European Pharmacopoeia chapter 2.6.7. Comparability studies for rapid methods intended to satisfy the US FDA should be discussed with that agency, as no formal guidance has been published.
What attributes should be considered when selecting a rapid mycoplasma detection method?
1. Sample volume. The current approved culture methods test at least 10 mL of sample. A rapid method intended to replace the current methods should ideally be able to test an equivalent volume of sample. It may be difficult to gain FDA approval for nucleic acid-based methods which can test only microliter amounts of sample.
2. Duration. Hybrid culture/PCR systems may take as long as 14 days to complete, while direct nucleic acid-based methods should be completed within a week or less.
3. Specificity. European Pharmacopoeia 2.6.7 specifies that the nucleic acid test must be able to exclude closely-related bacterial species.
4. Sensitivity. FDA indicates that the rapid method should be equivalent to or better than the approved culture method in terms of sensitivity (limit of detection), based on comparability studies using viable mycoplasma organisms.
5. Orthogonal endpoints. Having two or more orthogonal endpoints is desirable to allow one to discriminate between low level positive and negative signals.
6. Validation status. For contract methods, has the method been validated per European Pharmacopoeia 2.6.7? For kit methods, has the vendor validated the method per European Pharmacopoeia 2.6.7?
7. Drug Master File. For kit methods, has the vendor submitted a drug master file to the FDA for the method?
These considerations should help in deciding among the various options now available for implementing rapid nucleic acid-based mycoplasma testing for biopharmaceutical lot release applications.
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