The United States Pharmacopeia has recently published chapter <63> Mycoplasma Tests. Biopharmaceutical companies conducting mycoplasma testing as a lot release assay for unprocessed bulk material will need to comply with this new regulation once it becomes effective later this year. In this article, we compare the language of USP <63> to that of the existing regulation (European Pharmacopoeia chapter 2.6.7 Mycoplasmas) and guidance (1993 Points to Consider in the Characterization of Cell Lines used to Produce Biologics).
The United States Pharmacopeia's (USP) new chapter <63> Mycoplasma Tests(1) 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 (PTC) and the European Pharmacopoeia (EP) chapter 2.6.7 Mycoplasmas.(2–3) A separate regulatory document (21 CFR 610.30)(4) will not be considered here because the language contained therein is specific to mycoplasma testing of live and inactivated viral vaccines. Because there had been some methodological differences between the FDA guidance and EP 2.6.7, the industry was hoping that the new USP chapter would serve to harmonize mycoplasma testing as much as practically possible, and would not lead to additional points of discrepancy.
Chapter <63> was published in the USP 33/NF 28 issuance that became available on November 1, 2009. USP 33/NF 28 was originally scheduled to become effective on May 1, 2010, but was retracted because of monograph errors that were introduced during conversion to a new format. The announcement to that effect was posted on the USP website on January 12, 2010.(5) USP 33/NF 28 was re-issued in March 2010 with an official date of October 1, 2010.
COMPARISON OF USP <63>, 1993 PTC, AND EP 2.6.7
A quick look at the new USP chapter <63> indicates that the text and tables comprising that chapter were based in large part on EP 2.6.7, with only certain aspects being derived from the 1993 PTC. A comparison of the three documents (USP <63>, 1993 PTC, and EP 2.6.7) 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 and types of positive controls to be used. The substantive methodological differences between the new USP chapter <63>, EP 2.6.7, and the 1993 PTC are outlined in Table 1.
Nutritive properties are to mycoplasma testing as growth promotion is to sterility testing. What is being considered is the ability of the growth media (agar and broth) to support the growth and detection of mycoplasma organisms if present in the test inoculum. To assess the nutritive properties of various growth media, the EP and USP documents each mandate that no more than 100 colony forming units (CFU) of model mycoplasma species be inoculated into each batch of growth medium to be used in an assay. For interpretation of the results for solid media (agar plates), EP 2.6.7 states:
"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)."(3)
There is a different requirement within USP <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."(1)
Assuming an inoculum of exactly 100 CFU is used for an organism, the acceptable ranges for the recovery of that organism on agar plates would be 32–316 colonies for the USP version versus 20–500 colonies for the EP version. The USP version is therefore more stringent in this respect. The language for assessment of nutritive properties in liquid media (broth) is essentially the same for EP 2.6.7 and USP <63>. The interpretation of growth promotion (nutritive properties) in liquid and solid media is not discussed within the 1993 PTC or 21 CFR 610.30. The language within the 1993 PTC is as follows:
"Each lot of agar and broth medium should be free of antibiotics except for penicillin, and each lot of medium should be examined for mycoplasmal growth-promoting properties.(2)
The requirement in 21 CFR 610.30 also is very general:
"The media shall be such as have been shown to be capable of detecting known Mycoplasma and each test shall include control cultures of at least two known strains of Mycoplasma, one of which must be M. pneumoniae."(4)
Test materials being evaluated for the presence of infectious mycoplasma have the potential of inhibiting the growth of those organisms, thereby causing interference with the detection endpoint. Therefore, both EP 2.6.7 and USP <63> address the evaluation of inhibitory substances within the material to be tested for mycoplasma. The procedure used is similar in each case. No more than 100 CFU of model mycoplasma species are inoculated in the presence or absence of the test material into the solid and liquid media used in the mycoplasma detection assay. The interpretation of the results for solid media (agar plates) is analogous to that for the nutritive properties discussed above. EP 2.6.7 states that there are inhibitory substances in the test material:
"…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…" (3)
The USP <63> version indicates that there are inhibitory substances in the test material:
"… 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."(1)
USP <63> specifies a tighter acceptance range than EP 2.6.7, with the result that the USP version is again more stringent in this respect. The language addressing assessment of inhibitory substances for the liquid medium (broth) portion of the mycoplasma detection assay is essentially the same for EP 2.6.7 and USP <63>. The assessment of inhibitory substances in a test material is not addressed within the 1993 PTC or 21 CFR 610.30. Despite this, good science dictates that the possibility of inhibitory substances in a test material be evaluated during mycoplasma testing to avoid false negative results.
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"
In EP 2.6.7, this is defined as:
"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.
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.
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)
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/U... .
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.