Endotoxin Detection Methods for
Pharmaceuticals and Medical Devices
The Horseshoe Crab
a source of LAL reagent
- Endotoxins (aka pyrogens) are found in the cell walls of Gram -ve bacteria
- Heat stable
- Mammals can be severely affected by small doses
- also called Bacterial Endotoxin Test or BET
During the early days of the pharmaceutical
industry it was noticed that some solutions when injected into the bloodstream
induced fevers. Investigations found that almost all of these fevers were
associated with a group of contaminants termed pyrogens ( 'heat or fever
generating'). These were classified as either exogenous or endogenous pyrogens.
Exogenous pyrogens are fever causing materials found in the environment, of
these - endotoxins are the most researched and are lipospolysacchrides (LPS),
found in the outer membrane of the cell wall of Gram -ve microorganisms, they
are heat stable and can cause severe patient reactions when present in
parenterals or medical devices.
Endotoxin toxicity is not dependant upon a living cell, heat sterilization or
other chemical/physical processes are ineffective control measures as killing
the cells actually releases 'free' endotoxin from the cell wall.
As all mammals can be affected by endotoxins (although sensitivity levels vary)
one of the first tests used to determine if endotoxins were present, was the
Rabbit Pyrogen Test (RPT). A rabbit was inoculated with the test substance and
then monitored it to see if a fever was induced. This test however does not
give a quantitative result, is time consuming and is not suitable for products
that may in themselves adversely effect the animal.
The most commonly used approach now is a Limulus Amoebocyte Lysate or LAL test.
The LAL test focuses in particluar on 2-keto-3-deoxyoctonoic acid - and it is
this which is used as an indicator in the majority of endotoxin assays.
for these items:
LAL is a reagent derived from
the blood cells of the horseshoe crab, unlike a mammal the crab does not have a
developed immune system, however the LAL component in its blood will bind to
and inactivate endotoxins, in the crab the resulting clot also forms a
protective barrier against bacterial infection.
At its simplest the LAL test consists of adding LAL reagent to the sample in a
test tube, incubating at 37°C for 1 hour. The tube is then gently inverted
- if a gel or clot has formed then a positive result is recorded.
A variety of LAL assay options are available - gel-clot; turbidimetric; and
chromogenic measurement. The latter 2 can be measured either as an endpoint or
kinetic reaction. The primary requirement for all methods is that the test
reagents, containers, pipettes etc. are pyrogen free.
The Gel-clot is the simplest and most widely used, it is also fully
described by most pharmacopaeis. The method can be qualitative or
semi-quantitative when comparing a sample against a dilution series of an
endotoxin standard. Can be used to provide a pass/fail for a certain limit and
is best used with low sample numbers. The method may be performed manually with
little or no requirement for instrumentation.
Turbidimetric systems depend upon an increasing concentration of
insoluble coagulin released as the test progresses, this changes the turbidity
of the sample solution. The change in turbidity can be measured either as
transmission or absorbance requiring at least a spectrophotometer.
Chromogenic assays depend upon a chromogen which changes color if
endotoxin is present in the sample. The higher the concentration of endotoxin
then the more chromgen is released. Optical reading device operating at the
chromgen wavelength is required.
Both the turbidimetric and chromogenic assays can be performed either as
endpoint or kinetic assays.
Endpoint assays involve the measurement of the turbidity or color after
incubation at a fixed temperature and time period. Instrumentation requirements
can be more basic than those needed for kinetic assays.
Kinetic assays measure the rate of change in turbidity or color during the
assay. Instrumentation is used to incubate at a fixed temperature, take
measurements, compare those measurements and generate the results. Kinetic
assays can provide a greater sensitivity over a wider range than endpoint
assays but more sophisticated instrumentation is usually required.
Because the potency of an endotoxin to cause pyrogenic reaction will vary
according to the nature of the toxin, the FDA developed Endotoxin Units (EU)
for result comparisons.
Choice of LAL technique may be proscribed by the pharmacopaies but may also be
affected by other factors such as the inherent turbidity or color of the sample
as well as required sensitivities. Electronics technology has also advanced to
the stage that portable endotoxin systems with results available in 15 minutes
are now on the market, which are more suited for lower sample numbers with the
advantage of little operator training being required. If higher sample numbers
are to be processed more sophisticated instrumentation in the form of dedicated
systems or incubated microplate readers with appropriate software programs can
be used, automated robotic systems are also available.
Recently there have been developments intended to decrease the reliance on
horseshoe crabs. Examples of these Alternative methods include:
- genetically engineered recombinant factor C - the initial element of the LAL
clotting cascade is linked to fluorogenic substrates;
- cell lines sensitive to LPS linked to color changes in the test medium;
Although a postive LAL test indicates the presence of pyrogens a negative
result does not necessaryily mean the product is pyrogen free, as non-endotoxin
pyrogens may be present but not picked up by the LAL test. But this test does
enable products that are unsuitable for the rabbit test to be screened.