INCUBATION TEMPERATURES

Minor differences in incubation temperature ranges for test cultures exist between the various mycoplasma test documents (36 ± 1 °C for USP <63> and 1993 PTC, versus 35–38 °C for EP 2.6.7). In addition, there are some minor differences in the terminology used for the incubation condition for the solid media. For instance, this condition is referred to as microaerophilic in the EP 2.6.7 and USP <63>. Microaerophilic is defined in USP <63> as:

"nitrogen containing 5 to 10 per cent of carbon dioxide"

"hydrogen atmosphere containing <0.5% oxygen and/or nitrogen containing 5%–10% carbon dioxide in nitrogen."

In the 1993 PTC, this condition is simply referred to as:

"5 to 10 percent carbon dioxide in nitrogen and/or hydrogen atmosphere containing less than 0.5 percent oxygen,"

without invoking the terms microaerophilic or anaerobic.

POSITIVE CONTROLS

The USP <63> and 1993 PTC documents specify the number and types of positive controls to be used in the assays. For instance, the mycoplasma testing assay is to include at least two known mycoplasma species or strains as positive controls (one being a dextrose fermenter and the other an arginine hydrolyzer). On the other hand, EP 2.6.7 specifies that at least one of the six mycoplasma species listed in the chapter be used as a positive control.

PROVISION FOR NUCLEIC ACID-BASED TESTING

The new USP chapter <63> differs from EP 2.6.7 also in that the former does not address the requirements for validation of alternative methods for mycoplasma testing. The chapter does mention that alternative methods (nucleic acid and enzymatic) may be used, provided that these are validated and shown to be "comparable" to the existing culture (agar/broth and indicator cell) methods. Method development in recent years has occurred to the extent that there is now a choice of commercially available kits for conducting rapid mycoplasma testing. It appears that many of these, especially those based on quantitative polymerase chain reaction or transcription-mediated amplification, may be superior to the approved culture assay in specificity and sensitivity. The EP chapter 2.6.7 laid the foundation for use of nucleic acid-based mycoplasma detection tests for the first time in version 5.8 (effective July 2007). This provided the industry with regulatory expectations for implementing a rapid alternative test to the approved culture test for mycoplasma, which is 28 days in duration. A similar guidance has yet to be forthcoming from the FDA, and it appears that the USP also has chosen to leave the specifics of validation of these alternative methods to the FDA and EP.

SUMMARY

The new USP chapter <63> Mycoplasma Tests contains language derived from both the pre-existing EP 2.6.7 and 1993 PTC documents. The issue of inhibitory substances was not addressed within the FDA's 1993 PTC, so there are no conflicts in this respect between the PTC (per the above statement) and the new USP chapter <63>. Now, to be compliant with the FDA and EP 2.6.7 requirements as well as the new USP chapter, however, testing laboratories will have to make adjustment within their protocols to account for the stricter USP criteria for assessing nutritive properties and inhibitory substances. Once the new USP chapter becomes effective, the mycoplasma test will be considered compendial, which means that laboratories performing the mycoplasma test as described in the chapter will not be required to validate the method. The laboratories will only be required to perform verification (matrix qualification) for each sample type to be tested per USP <1226>.⁶

Raymond W. Nims, PhD, is a biosafety consultant at RMC Pharmaceutical Solutions, Inc., Longmont, CO, 301.639.9747, rnims@rmcpharma.com
and Elizabeth Meyers is a compliance manager at Amgen, Inc., Thousand Oaks, CA.

REFERENCES

1. US Pharmacopeial Convention (USP). USP 33/NF 28 <63> Mycoplasma tests. Rockville, MD; 2010.

2. US Food and Drug Administration. US FDA points to consider in the characterization of cell lines used to produce biologicals. Rockville, MD; 1993. Available from: http://www.fda.gov/downloads/BiologicsBloodVaccines/SafetyAvailability/UCM162863.pdf.

3. European Pharmacopoeia. EP 2.6.7 Mycoplasmas, 6th ed. Strasbourg, France; 2010.

4. Test for Mycoplasma. 21 CFR Sect 610.30.

5. USP. Public notices. Rockville, MD; 2010. Available from: http://www.usp.org/USPNF/jan12Recall.html.

6. USP. USP 33/NF 28. <1226> Verification of compendial procedures. Rockville, MD; 2010.