APR September/October 2020 - 102

MICROBIOLOGY

Is a Difference in LAL Reagent
Results Possible?
Yes. The LAL reagents are derived from horseshoe crabs and are
therefore of biological origin. It has been described that the lysate
is a relatively crude mixture and is not a single purified enzyme. This
means that the enzyme activity cannot be determined exactly for each
lot of lysate manufactured. Furthermore, the manufacturing process
includes the addition of buffers and detergents which contribute a
further source of variability.1 A reduced variability can be achieved by
using recombinant Factor C reagents.

Can a Variation in the Standard Curve
Effect the Test Result?
Yes. To quantify bacterial endotoxin, a standard curve is prepared in
order to determine the endotoxin activity of a sample. Therefore, the
quality of the standard curve is the basis of quantification. Using a
linear standard curve, a change of only 1% in y-intercept can result in a
change of up to 35% in measured endotoxin activity.1
In Figure 1, Y-intercept (Y-Achsenabschnitt) as a function of number of
analysis (Analysennummer) from trending analysis is shown. All data
points (full diamonds) fulfill the standard acceptance criteria. However,
the typical observed variations may lead to increased/decreased test
results depending on the y-intercept as small variations can lead to
relative high variations in measured EU/mL.

are ultimately biological assays, the lysates are intrinsically variable.2
Moreover, recent challenges like LER and the implementation of
recombinant tests brought up again discussions about Naturally
Occurring Endotoxins (NOE). Advocates of NOE in the field of LER are
refusing NOE when it comes to the comparison of test methods. It has
been stated that NOE more closely mimics a real life contamination
event,3 but on the other hand it has been communicated that NOEs
grown in laboratory are not representative of what occurs in nature.
This contradictoriness clearly reflects the incongruous application of
undefined endotoxin spikes during testing.

Can a Sample Composition Alter the
Detectability of Endotoxin?
Yes. There have been a lot of publications about Low Endotoxin
Recovery (LER) and endotoxin masking which can lead to
underestimation of endotoxin contents.2,4-6 Due to the presence
of certain excipients or active pharmaceutical ingredients or
combinations thereof, endotoxin can be masked. An example for the
detectability of endotoxin in a typical LER matrix is given in Table 1.
Thereby the detectability decreases although the endotoxin is not
degraded and potentially hazardous.
In order to reveal these effects so called LER studies are mandatory.
Table 1. Detection of endotoxin over time in a typical LER matrix
Low Endotoxin Recovery Study

[EU/mL]

M1 Time Point 0 days

64.0

M2 Time Point 1 days

27.8

M3 Time Point 2 days

17.6

M4 Time Point 3 days

7.3

M5 Time Point 7 days

4.8

The data is sourced from Low Endotoxin Recovery - Masking of Naturally Occurring Endotoxin6

Therefore, undiluted samples are spiked with endotoxin and held for a
certain period of time. More guidance for LER including strategies for
demasking is found in the Technical Report No. 82 from PDA.

Figure 1. Y-intercept of standard curves from trending analysis

Is Reference Standard Endotoxin Still
Representative for BET?
Yes. Reference Standard Endotoxin (RSE) is the benchmark and allows
comparability of test methods. Due to the heterogeneity of endotoxin,
standardization of bacterial endotoxin tests was very challenging
in the early time of BET. Only the introduction of RSE was the key
factor to control the quality of BET, since Limulus-based approaches

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Do (13)-ß-D-Glucans Affect the
Endotoxin Test Result?
Yes. The LAL test includes per se the Factor G reaction pathway which
is described to react with (13)-ß-D-glucans.7 This reaction pathway
has been identified years ago. Interestingly this pathway is unequally
pronounced in different LAL tests. There are also agents available to
repress Factor G reaction pathway. Unfortunately, it is neither proven
that a) all glucans nor b) that their full activity is blocked. Obviously,
glucans are very heterogeneous and present in various aggregation
states and can be derived from a variety of sources. Once present in a
sample the absolute differentiation between LPS and glucans with LAL
is virtually impossible.

| September/October 2020

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APR September/October 2020

Table of Contents for the Digital Edition of APR September/October 2020