USP <63> Mycoplasma Tests: A New Regulation for Mycoplasma Testing - What you need to know about USP chapter . - BioPharm International

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USP <63> Mycoplasma Tests: A New Regulation for Mycoplasma Testing
What you need to know about USP chapter <63>.


BioPharm International
Volume 23, Issue 8

NUTRITIVE PROPERTIES

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

INHIBITORY SUBSTANCES

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.


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