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.

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. 

Qualitative: Simple Yes/No Answer.

• Gel Clot LAL Assays:
  Simple LAL test. Visual inspection of gel formation; does not require incubating reader and software.

Quantitative: Reveals not only the presence of endotoxin, but also the amount present. Results calculated from a standard curve.

• Chromogenic LAL Assay:
  Endpoint and kinetic. Kinetic or endpoint measurement of color development allow a quantitative readout of the endotoxin concentration in the sample measured.


• Turbidimetric LAL Assay: 
  Kinetic measurement of turbidity development is a cost-effective way of BET testing water samples or large volume parenterals.


• Recombinant Factor C: 
  Non-animal alternative to LAL based on the recombinantly produced form of Factor C, the first component in the horseshoe crab clotting cascade. Does not rely on horseshoe crab blood as the source testing reagent.

Note that any of Lonza’s quantitative testing products can be automated for higher throughput testing needs. Learn more about endotoxin automation.

To learn more about the different endotoxin testing methodologies, access our On-demand Webinar, "Endotoxin Detection: Which Method Is Best for Me?" Log in or create a Lonza website account to view. 

Vaccination is now a common and successful method of disease prevention and mitigation around the world. As an injectable product, there is a danger of endotoxin contamination, which could have serious health risks to the person receiving the vaccine. In the United States, such products are regulated under the authority of the Food, Drug, and Cosmetic Act by the U.S. Food and Drug Administration. The regulations may be found in Title 21 of the U.S. Code of Federal Regulations. Under 21 CFR 211.167 it states:

Sec. 211.167 Special testing requirements.
(a) For each batch of drug product purporting to be sterile and/or pyrogen-free, there shall be appropriate laboratory testing to determine conformance to such requirements. The test procedures shall be in writing and shall be followed.

Vaccines are regulated as a biological by FDA’s Center for Biologics Evaluation and Research (CBER). All biological products are required to be tested for pyrogenicity, unless the product is specifically exempted by regulation. Under Part 610 General Biological Products Standards, it states:

Sec. 610.13 Purity.
(b) Test for pyrogenic substances. Each lot of final containers of any product intended for use by injection shall be tested for pyrogenic substances by intravenous injection into rabbits as provided in paragraphs (b) (1) and (2) of this section: Provided, That notwithstanding any other provision of Subchapter F of this chapter, the test for pyrogenic substances is not required for the following products: Products containing formed blood elements; Cryoprecipitate; Plasma; Source Plasma; Normal Horse Serum; bacterial, viral, and rickettsial vaccines and antigens; toxoids; toxins; allergenic extracts; venoms; diagnostic substances and trivalent organic arsenicals.

As an exempted biological, viral vaccines need not be tested in rabbits for pyrogenicity for several reasons. First, there is a possibility that the vaccine itself is incompatible with rabbits, causing the rabbit to die. Secondly, vaccines are meant to stimulate the immune system, and cause a fever in the animal unrelated to endotoxin contamination. Therefore, a different analysis method for pyrogenicity must be used, such as the Limulus Amebocyte Lysate (LAL) Assay, and performed as per the United States Pharmacopeia (USP) General Chapter <85> Bacterial Endotoxins Test.