GMP News: Question and Answers to Endotoxin Masking and Low Endotoxin Recovery (LER)

 Question
and Answers to Endotoxin Masking and Low Endotoxin Recovery (LER)

 

Masking
of Endotoxins and LOW Endotoxin Recovery is a currently often discussed topic
related to the quality of pharmaceutical products. As part of the webinar on
Endotoxin Masking and Low Endotoxin Recovery (LER) end of September the
participants had a number of questions. The speaker, Johannes Reich, answered
these questions at the end of the webinar. Following you will find a summary of
these questions and answers.

"The
following questions and answers are a summary of the points raised in the
webinar. The provided answers are not binding and shall only serve for
guidance.

Do
studies for time-dependent endotoxin recovery have to be conducted for all
formulations?

Yes,
it is not sufficient to merely test single substance groups or formulation
classes, since endotoxin masking depends on numerous different factors. For
instance, different pH settings may significantly influence masking under
otherwise identical conditions.

What
is the difference between "test interference" and "endotoxin
masking"?

Both
phenomena have different underlying mechanisms. In case of "test
interference", the detection system/ the enzymatic reaction is directly
disturbed by the ambient conditions. For example, protease inhibitors can
impair the detection enzyme and thereby cause a false-negative result.

On
the other hand, if "endotoxin masking" occurs, the endotoxin to be
detected itself is influenced by the ambient conditions. This means that the
active form (supramolecular structure) of the endotoxin has changed, leading to
lower activities that are detected. The enzymatic detection reaction is however
not inhibited in this case. In practice, both phenomena may occur
simultaneously.

How
can you differentiate between interference and masking?

As
interference is time-independent and masking is time-dependent, time-dependent
experiments are required in addition to the positive control. This means that
the recovery rate decreases over time, if masking occurs. To differentiate
between the phenomena, a sufficiently high spike concentration has to be chosen
for the undiluted samples to later dilute for in turn excluding test
interferences.

How
long and at which incubation temperature shall a masking experiment be
conducted?

The
length of incubation is highly dependent on the respective sample. A
significant decrease of endotoxin recovery (<50%) has been observed in some
cases already after a few minutes, e.g. 30 min. On the other hand, there have
been cases where masking was only found after several days, e.g. 5 days.

The
incubation temperature shall be chosen to match the sample's ambient
temperature which commonly occurs in the course of its handling.

Which
value is used as start in a masking kinetic study?

The
endotoxin recovery in LRW (LAL reagent water) can be used as start value
(100%).

Which
concentration shall the endotoxin spike have?

The
optimum spike concentration depends on the sample to be analyzed and shall be
adjusted to the endotoxin limit to be detected and the MVD (maximum valid
dilution). Low spike concentrations, e.g. <10 EU/ml, may even happen to
behave unfavorably in pure LRW.

How
do you ensure that LRW and/or the sample tube do not mask endotoxin?

To
exclude endotoxin masking in LRW or by the sample tube, the endotoxin spike (in
a single sample tube) shall be monitored together with the masking sample over
time.

Which
test systems can be used for detection?

All
common detection systems can be used for conducting endotoxin masking
experiments, e.g. LAL (turbidometric, chromogenic etc.), recombinant detection
systems or monocyte activation tests (MAT).

Which
measures shall be taken after observing masking?

After
observing masking, the optimization of the used test system, e.g. with respect
to sample preparation, or the replacement with an alternative method are
advised.

What
is the principle of demasking?

It
is assumed that the detectability of endotoxin depends on its supramolecular
structure. This structure may exhibit various aggregation forms and thus
different activities depending on the ambient conditions. In the course of
masking, the endotoxin structure is converted into an undetectable state, e.g.
monomers. To return masked endotoxin into an active state, the ambient
conditions have to be chosen in a way that shifts the balance back toward
"self organization" into active aggregation forms."

Johannes
Reich, Experts from Industry and Representatives of the European Authorities as
well as of the FDA will present the current developments at the Endotoxin and
Pyrogen Testing Conference on 19 and 20 November in Düsseldorf/Neuss, Germany.

GMP News: Question and Answers to Endotoxin Masking and Low Endotoxin Recovery (LER)