The Bacterial Endotoxin Test (BET) is a specific in-vitro test developed for the identification and quantification of bacterial endotoxins, which is a part of the gram-negative bacteria cell wall. BET is conducted as part of a lot release test for medical devices with direct or indirect exposure to the cardiovascular system, lymphatic system, or cerebrospinal fluid.
Injectable pharmaceutical products should also be screened for bacterial endotoxins. Routine monitoring of water systems and incoming materials can help to ensure that endotoxins do not contribute to the final product.
The Importance of Testing
Bacterial endotoxins are ubiquitous in nature and pose a threat to manufacturers of all parenteral drugs and medical devices. Not only are they environmentally friendly everywhere, but they are also very difficult to remove once they are introduced into the finished parenteral product.
For example, sudden infant death syndrome has been linked to the changing levels of bacterial endotoxin in the bloodstream.
The introduction of these molecules can come from simple contact with contaminated parenteral devices or drugs. Ensuring that endotoxin-free instruments and medications are available is especially critical in the treatment of vulnerable patients, including the elderly, intensive care patients, and children.
In the early days of injectable pharmaceutical materials, there was no method developed for testing for pyrogenic contaminants. If a patient received an injection, there was a high likelihood that they would spike a fever or ‘injection fever’.
In the 1970s, pharmaceutical companies experimented with the LAL test and compared it with the RPT, found that the LAL test was more accurate and sensitive (4), and provided cost-effectiveness to companies as there was no need to establish rabbit colonies for product testing.
After its launch, the LAL test has become one of the most important methods used by the pharmaceutical industry. It required a degree of bacteria and pyrogen monitoring that eluded manufacturers when the RPT was the only pyrogen test available. The LAL test ensured the absence of pyrogens in raw materials, water for injection systems, in-process samples, and finished goods.
Methods:
There are currently three compendial bacterial endotoxins (BET) tests, namely, gel-clot, kinetic chromogenic, and kinetic turbidimetric tests that can be used to assess injectable pharmaceutical products and implantable medical devices for commercial release.
- The LAL Gel-Clot Test. This form is the oldest and the easiest of the three. The key advantage of the LAL gel clot test is its simplicity as it is easy to understand and can be done without costly equipment. The gel-clot assay works by forming a clot in the presence of endotoxins by clotting the coagulogen to coagulin.
This method can be conducted either as a limit test or as a semi-quantitative test, giving a positive or negative effect on the sensitivity of the lysate used. Dilutions are evaluated with lysate and the endpoint is used to measure the estimated amount of endotoxin.
Despite the benefits of the process, such as dealing with fewer components, resulting in a lower probability of outcomes being compromised by inhibition or enhancement of the sample, some drawbacks have hindered its popularity.
The time taken to prepare samples using this method has made it less common for use in raw material research in some laboratories. Besides, the sensitivities of lysate used in the gel clot process are less sensitive than quantitative methods.
- Kinetic turbidimetric LAL test (KTA). KTA is one of the quantitative assays in which the absorption versus endotoxin concentration calculation is used to produce a standard curve.
During the LAL-endotoxin reaction, the solution mixture is gradually turbid as the gel starts to form. The time needed for these changes in turbidity to occur is inversely proportional to the amount of endotoxin present in the sample (i.e., more endotoxin requires less time).
The endotoxin in the sample can be calculated by comparing the time it takes to reach a given turbidity level with the times obtained from a series of endotoxin standards (standard curve).
Sensitivity can be higher with KTA over the gel clot process, and while KTA can be performed in test tubes, it is more common to conduct tests on plastic, 96-well microtiter plates.
The spectrophotometers used to calculate changes in turbidity can be captured using appropriate software and the assimilated data is used to produce quantitative reports. The same data can be submitted to the LIMS database for monitoring and trending.
The most important benefits of quantitative approaches over the gel-clot approach are improved sensitivity and the ability to extrapolate findings. It can be useful to be able to extrapolate quantitative data when analyzing raw materials because it can provide insight into possible sources of endotoxin contamination.
- Chromogenic substrate test. In the third method, The more endotoxin, the quicker the substrate is glued, resulting in a darker yellow hue. These chromogenic tests have an elegant technique and have many of the advantages that KTA has, such as improved sensitivity and extrapolation of quantitative data.
However, chromogenic experiments are more costly. Chromogenic substrate content requires months of synthesization and, as such, is very costly to produce.
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