The United States Pharmacopeia’s (USP) new chapter <63> Mycoplasma Tests was planned to become effective on May 1, 2010 as part of USP 33. This new chapter was intended to fill a void in the USP for mycoplasma testing, which had been addressed previously within the FDA’s 1993 Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals and the European Pharmacopoeia (EP) chapter 2.6.7 Mycoplasmas (a separate document applying only to mycoplasma testing of live and inactivated viral vaccines, 21 CFR 610.30 will not be considered here). Since there were some methodological differences between the FDA guidance and the EP chapter, it was hoped by the industry that the USP guidance would serve to harmonize mycoplasma testing as much as practically possible.
Indeed, a quick look at the new USP chapter <63> indicates that the chapter was based in large part on EP chapter 2.6.7. A comparison of the three documents (USP, FDA, and EP) reveals methodological differences in only a few areas. These include the assessment of nutritive properties of the solid growth media (agar) used for mycoplasma testing, the assessment of inhibitory substances in the test material, the incubation temperature ranges to be used, and the number of positive controls to be used.
The EP chapter 2.6.7 states that “The solid medium complies with the test if adequate growth is found for each test micro-organism (growth obtained does not differ by a factor greater than 5 from the value calculated with respect to the inoculum)”. There is a different requirement within USP chapter <63>: “The solid medium complies with the test if a count within a 0.5-log unit range of the inoculate amount is found for each test microorganism”. Assuming an inoculate of 100 colony forming units (CFU), the acceptable ranges for the recovered organisms would be 32-316 CFU for the USP version vs 20-500 CFU for the EP version. The USP version is therefore more stringent in this respect.
Similarly, for the assessment of inhibitory substances, EP chapter 2.6.7 states that “…if plates directly inoculated with the product to be examined have fewer than 1/5 of the number of colonies of those inoculated without the product to be examined” there are inhibitory substances in the test material. The USP version indicates that there are inhibitory substances “…if plates directly inoculated with the test article/material are not within a 0.5-log unit range of the number of colonies of those inoculated without the test article/material.” So the USP version is again more stringent in this respect.
Minor differences in incubation temperature for test cultures exist between the documents (36 ± 1°C for the USP and PTC documents vs 35-38°C for the EP chapter). The USP and PTC documents specify the number and types of positive controls to be used in the assays: at least two known Mycoplasma species or strains should be included as positive controls (one a dextrose fermenter and one an arginine hydrolyzer). The EP chapter specifies that at least one of the six Mycoplasma species listed in the chapter be used as a positive control.
The USP chapter <63> differs from EP chapter 2.6.7 also in that the former does not provide requirements for validation of a nucleic acid-based test for mycoplasma. The USP chapter mentions the possibility of replacing the culture method with an alternative (nucleic-acid or enzymatic) method, stating that the alternative method must be validated and shown to be comparable to the agar/broth and cell culture methods. The EP chapter laid the foundation for validation of a nucleic acid-based mycoplasma detection test for the first time in version 5.8 (effective July 2007). This provided the industry with expectations for implementation of a rapid alternative test to the approved culture test for mycoplasma, which is 28 days in duration. Similar guidance is not yet forthcoming from the FDA or USP.
The issues of nutritive properties and inhibitory substances are not addressed within the FDA’s 1993 Points to Consider guidance. In order to now be compliant with the FDA and EP requirements as well as the new USP chapter, testing labs will have to make adjustment within their protocols to account for the stricter USP criteria for assessing nutritive properties and inhibitory substances. Due to errors within some of the monographs to appear in the issuance of the USP to become effective May 1, 2010, this issuance of USP 33, including chapter <63>, was retracted in January of 2010. However, it will be re-issued in March 2010 with an official date six months after reissue, and the methodological differences may need to be accounted for in testing protocols used by quality control laboratories for which USP compliance is applicable.
Once this chapter becomes effective, the mycoplasma test methods described will be considered compendial, meaning that labs following the methods outlined in the chapter will not be required to perform method validation. The labs will only be required to perform method verification for each test sample type (matrix qualification) per USP <1226>.
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