APR Sept/Oct 2023 - 51

days. Although it is possible to treat patients with such products before the result of
the culture-based test becomes available, this is not the ideal situation as freedom from
mycoplasmas is part of a products' safety profile.
Alternative nucleic acid methods save time
For the above reasons many facilities have turned to using rapid enzymatic activitybased
methods or nucleic acid amplification techniques, in particular PCR, preferring to
put up with the validation effort that implementing such alternative methods require
to avoid the culture method wherever possible. Equivalency of the alternative method
to the compendial culture-based method is demonstrated using a performance
comparison that includes sample suitability, robustness, sensitivity, specificity and the
ability to detect a broad spectrum of species. There are, as yet, no specific documents
on the design of such comparative studies for an FDA review but some guidance can
be taken from Ph. Eur. 2.6.7. It should be noted that validation of a direct PCR method
without culturing can be challenging as there tends to be a relatively high risk of false
negative and false positive results.
However, alternative methods are not making cultivation obsolete. Cultivation is
regularly required for validation purposes. In addition, to achieve a suitable limit of
detection of below 10 CFUs/mL or if the sample volume is below 1 mL, rapid methods
such as PCR may need a prior enrichment step. Furthermore, inhibitory substances
can impact rapid methods, with dilution by enrichment a possible remedy. In cases
where the sample matrix renders direct PCR testing unreliable, centrifugation to clear
the matrix has been a popular alternative to enrichment but will probably cease to be
an option when the next revision of Ph. Eur. 2.6.7 comes into force. The reason is that
mycoplasmas have the tendency to adhere to mammalian cells, so pelleting away the
eukaryotic cells and debris will also remove bacteria from the sample, which can lead
to false-negative test results. Nevertheless, rapid methods to test for mycoplasmas have
resulted in valuable time savings for biologics' manufacturers who have succeeded with
implementation.
Ready-to-use
Culture Media
for Mycoplasma
Testing
Now with Extended
Shelf Life
Friis, Frey, and Hayfl ick
media available in both
liquid and solid.
SigmaAldrich.com/mycoplasma
Find out about our full range of ready-to-use solid and liquid
media for compendial mycoplasma testing.
MilliporeSigma is the U.S. and Canada Life
Science business of Merck KGaA, Darmstadt,
Germany.
© 2023 Merck KGaA, Darmstadt, Germany and/or its affiliates.
All Rights Reserved. MillipreSigma, the vibrant M, and Millipore
are trademarks of Merck KGaA, Darmstadt, Germany or its
affiliates. MAS-100 instruments manufactured by MBV AG,
Switzerland, www.mbv.ch, MBV. Air. Nothing else. All other
trademarks are the property of their respective owners.
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APR Sept/Oct 2023

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