Product analysis - Pharmaceutical Biotechnology: Concepts and Aapplications - page 195

• subclinical infection/poor overall animal health can also lead to false positive results;

• use of different rabbit colonies/breeds can yield variable results.

Another issue of relevance is that certain biopharmaceuticals (e.g. cytokines such as 1L-1 and
TNF; Chapter 9) themselves induce a natural pyrogenic response. This rules out use of the rabbit-
based assay for detection of exogenous pyrogens in such products. Such diffi culties have led to the
increased use of an in vitro assay; the Limulus ameobocyte lysate (LAL) test. This is based upon
endotoxin-stimulated coagulation of amoebocyte lysate obtained from horseshoe crabs. This test
is now the most widely used assay for the detection of endotoxins in biopharmaceutical and other
pharmaceutical preparations.
Development of the LAL assay was based upon the observation that the presence of Gram-
negative bacteria in the vascular system of the American horseshoe crab, Limulus polyphemus ,
resulted in the clotting of its blood. Tests on fractionated blood showed that the factor responsi-
ble for coagulation resided within the crab's circulating blood cells, i.e. the amoebocytes. Fur-
ther research revealed that the bacterial agent responsible of initiation of clot formation was
endotoxin.
The endotoxin molecule activates a coagulation cascade quite similar in design to the
mammalian blood coagulation cascade (Figure 7.8). Activation of the cascade also requires
the presence of divalent cations such as calcium or magnesium. The final steps of this path-
way entail the proteolytic cleavage of the polypeptide coagulogen, forming coagulin, and a
smaller peptide fragment. Coagulin molecules then interact non-covalently, forming a 'clot'
or 'gel'.
The LAL-based assay for endotoxin became commercially available in the 1970s. The LAL
reagent is prepared by extraction of blood from the horseshoe crab, followed by isolation of its
amoebocytes by centrifugation. After a washing step, the amoebocytes are lysed and the lysate
dispensed into pyrogen-free vials. The assay is normally performed by making a series of 1:2
dilutions of the test sample using (pyrogen-free) WFI (and pyrogen-free test tubes; see later). A
reference standard endotoxin preparation is treated similarly. LAL reagent is added to all tubes,
incubated for 1 h, and these tubes are then inverted to test for gel (i.e. clot) formation, which would
indicate presence of endotoxin.
More recently, a colorimetric-based LAL procedure has been devised. This entails addition to
the LAL reagent of a short peptide, susceptible to hydrolysis by the LAL clotting enzyme. This
synthetic peptide contains a chromogenic tag (usually para -nitroaniline, pNA) which is released
free into solution by the clotting enzyme. This allows spectrophotometric analysis of the test sam-
ple, facilitating more accurate end-point determination.
The LAL system displays several advantages when compared with the rabbit test, most
notably:

• sensitivity - endotoxin levels as low as a few picograms per millilitre of sample assayed will be

detected;

• cost - the assay is far less expensive than the rabbit assay;

• speed - depending upon the format used, the LAL assay may be conducted within 15-60 min.