APR Sept/Oct 2023 - 50

QC CORNER
The Intricacies of Testing for Mycoplasmas
in Cell Culture Systems
Tim Cser
Senior Technology Specialist
MilliporeSigma, CA
An affiliate of Merck KGaA, Darmstadt, Germany
Mycoplasma contamination is a widespread and
reoccurring problem in many cell and tissue culture
systems that are designed to produce biologics such as
recombinant proteins, monoclonal antibodies and cell
therapeutics. These ubiquitous bacteria can grow to
high titers in culture media without exhibiting typical
bacterial contamination signs such as turbidity or pH
change. Their effects on cultured cells include altered
metabolism, slowed proliferation and chromosomal
aberrations, potentially compromising the quality of
the therapeutic and thus patient safety. This is why it is
pivotal to verify that cell cultures for the manufacture of
pharmaceuticals are, and remain, contamination-free.
Unfortunately, compendial sterility testing is unsuitable
to detect mycoplasmas as they are too small to be filtered
and will not grow in the tryptic soy broth (TSB) or fluid
thioglycollate medium (FTM) used for this test. For these
reasons there is dedicated regulation for mycoplasma
testing, most notably USP <63> and Ph. Eur. 2.6.7, with
guidance also given by CBER/FDA.
Based on a risk assessment, the manufacturer determines
at which stages of the production process to test for
mycoplasmas. Biopharmaceutical facilities that use
eukaryotic cells for the production of vaccines have to
test their cell banks and virus seed lots as well as perform
in-process testing, particularly on the bulk vaccine
Yoann Gatineau
R&D Technical Leader
Millipore SAS, Molsheim, France
An affiliate of Merck KGaA, Darmstadt, Germany
during harvest. End product tests usually do not yield as
meaningful results because the bacteria would typically
be less concentrated by this stage.
The complex compendial
culture method
For many stages of the production process, compendial
mycoplasma testing
involves both a culture media
test to detect cultivable strains and an indicator cell
test for fastidious, non-cultivable strains, which is less
sensitive and requires highly skilled staff to perform.
The compendial culture media method is a painfully
complex and long-drawn-out process. It requires up to
three different media, Hayflick, Frey and Friis, that are
specially formulated to jointly cover the detection of a
broad spectrum of Mycoplasma species as well as related
Mollicutes bacteria such as Acholeplasma laidlawii. These
media are complicated to make in a lab due to some
sensitive components. The shelf lives are also short,
although we at MilliporeSigma, the only major supplier
worldwide, have recently validated a longer four months
for our agar and six months for our broths. The culture
method also requires a relatively high volume of test
sample and, most significantly, takes 28 days to deliver
results-much too long for many modern-day biologics
such as stem cell and regenerative medicine products,
which have to be administered to patients within a few
50 |
| September/October 2023
APR Sept/Oct 2023

Table of Contents for the Digital Edition of APR Sept/Oct 2023
