An Introduction to Bacterial Endotoxin Testing

The presence of Lipid-A in the bloodstream promotes the expression of proinflammatory cytokines and tissue factor by endothelial cells, leading to cell apoptosis. If it is released in sufficiently large quantities, Lipid-A causes the toxic effects associated with endotoxin infection in patients, including fever, diarrhea, vomiting, and possibly fatal endotoxic shock (septic shock).

 

Bacterial endotoxin is one of the most dangerous and common pyrogenic (fever-inducing) contaminants of parenteral pharmaceutical products, for four main reasons:

1. It is ubiquitous in nature (Gram-negative bacteria can proliferate in nothing more than clean water)
2. It has potent toxicity
3. It is stable under extreme conditions
4. It is likely to occur in the manufacturing process 

 

Reliable and efficient testing for the presence of bacterial endotoxins is therefore essential in the pharmaceutical and biomedical industries. Any injectable or implantable products labeled as pyrogen-free or sterile must undergo endotoxin testing before release. This protects patients from harmful endotoxin poisoning, and facilitates compliance with regulatory guidelines and cGMP. The most well-established and widely used bacterial endotoxin test is the LAL assay.

Mitigating risk to patients and complying with regulatory standards is vital in the pharmaceutical and biomedical industries. One crucial aspect of risk management is to ensure that any parenteral medicines and implantable devices released to the market are not contaminated with harmful bacterial endotoxins. As an essential requirement of current Good Manufacturing Practices (cGMP) and other quality control regulations, many companies use LAL testing to detect the presence of endotoxins in their products throughout the manufacturing processes. Often, companies will also test the raw materials for endotoxins before manufacturing begins, to meet Quality by Design criteria and assure low or negligible endotoxin content in raw material if possible.

In the US, the LAL assay relies on a reagent made from the harvested blood of wild Atlantic horseshoe crabs (Limulus polyphemus) that inhabit eastern coastal areas. In south-eastern and eastern Asia, three other horseshoe crab species (Tachypleus spp.) are harvested for their blood to produce another type of BET reagent, known as TAL. However, the TAL reagent is used in Asia, while LAL is used worldwide.

Even minimal amounts of endotoxin, less than a billionth of a gram, can trigger this immune response. This occurs via a complex clotting cascade, which has been extensively investigated since the LAL assay was first developed. The first enzyme in this cascade is the Factor C enzyme (the biosensor), which binds to the hydrophobic Lipid A component of the LPS molecule to initiate a cascade of enzymatic reactions that result in the formation of a blood clot. LAL testing takes advantage of this endotoxin-sensitive clotting cascade to produce a qualitative assessment of the presence/absence of endotoxin in a sample. Since it was first developed, numerous attempts have been made to improve the sensitivity and efficiency of the LAL assay by changing the formulation of the LAL reagent and the assay methodology.

This has raised serious challenges for horseshoe crab conservation. One key issue to the endotoxin testing community is that, as global populations rise, the pharmaceutical and biomedical industries will need to serve growing healthcare markets. This will increase demand for LAL, potentially jeopardizing sustainable harvesting practices and threatening already depleting horseshoe crab populations. These considerations have contributed to the recent development of alternative BET methods that do not rely on harvested crab blood: the recombinant Factor C (rFC) assay.

Four main types of bacterial endotoxin testing methods have been developed based on the principles of LAL testing. These all have important applications in quality control (QC) testing during the manufacture of parenteral medicines and injectable devices. Turbidimetric, chromogenic and rFC assays are quantitative, revealing not just the presence of endotoxin but also the amount present wheras the gel clot assay offers a simple but qualitative yes/no answer. Find out more about these different endotoxin testing methodologies by registering or logging in to watch our complimentary webinar “Endotoxin Detection – Which Method is Best for Me?”.