Pyrogen and bacterial endotoxin testing

BET and test types

The bacterial endotoxin test (BET) is a critical part of quality control (QC) testing. Testing products for the presence of bacterial endotoxins is a fundamental safety requirement in the pharmaceutical and biomedical industries and is performed on raw and in-process materials and for the final release of injectable or implantable products. These QC tests must comply with regulatory requirements enforced by global regulatory agencies.

The Limulus amebocyte lysate (LAL) assay was first developed in the 1960s and commercialized as a BET in the U.S. in the 1970s. The LAL assay is formulated using specialized blood cells, or amebocytes, obtained from the blue blood of Atlantic horseshoe crabs. The amebocytes function as the crab’s only immune defense: a blood coagulation system. After encountering foreign substances including endotoxin, amebocytes generate clots that immobilize and kill the pathogens.

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 (FC) 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 response to produce a BET assay that is reliable, sensitive and specific.

As LAL testing relies on harvesting the blood of wild horseshoe crabs, the conservation of horseshoe crab populations is a key priority. In the U.S., a variety of conservation initiatives have been widely successful in ensuring sustainable harvesting practices. However, the situation is more serious in Asia, where unsustainable blood harvesting practices for TAL production are causing serious population declines.

These considerations have contributed to the development of alternative BET methods that do not rely on harvested crab blood such as the recombinant Factor C (rFC) assay. These assays utilize a cloned version of the Factor C enzyme. When activated in the presence of endotoxin, Factor C cleaves a fluorescent substrate creating a signal that is measured in the rFC assay.

In total, four main types of BET methods have been developed based on the principles of LAL testing. They all have important applications in QC testing during the manufacture of parenteral medicines and injectable devices.

Test types

Lonza offers semi-qualitative tests that measure gel clot formation, such as the PYROGENT^® Gel Clot Assay, and quantitative tests that measure turbidity with the PYROGENT^® 5000 Kinetic Turbidimetric Assay, a color change with the Kinetic-QCL^® Kinetic Chromogenic LAL Assay or a fluorescence change with the sustainable PyroGene^® rFC Assay.

All of our quantitative tests are developed and optimized for use with the WinKQCL^® Endotoxin Detection Software and our incubating microplate readers. Further, all of our quantitative assays can be automated with our PyroTec^® PRO Automated Endotoxin Testing System.

Quantitative methods rely on the combination of test kits, validated instruments and software that provides data integrity. Download our Complete Testing Solutions e-book to learn about all of the kits, components, instruments and software that Lonza provides to meet all of your testing needs.

What is low endotoxin recovery (LER)?

Low Endotoxin Recovery (LER) is a controversial topic that has been circulating throughout the endotoxin detection community since 2013. This phenomenon is hypothetically described as a “masking effect” manifested in the biophysical formation of a complex that blocks the ability of Factor C, the main component in LAL detection, to bind endotoxin.

Two common drug excipients, polysorbate and citrate, have been identified as probable causes of the masking effect more commonly referred to as LER. These substances are estimated to be used in more than 70% of protein formulations. There is also some evidence that phosphate-containing formulations may also be affected by LER. However, the LER effect has only been observed in combination formulations of the aforementioned excipients, and not in individual raw materials.

Key points

• This 'phenomenon' has been observed for more than 20 years, but never identified
• Underlying mechanism of action is still unknown
• Primarily affects formulations of polysorbate/citrate buffers
• Naturally occurring endotoxin (NOE) preparations and other 'de-masking' solutions are currently being evaluated

Testing of biologics, including vaccines

Due to the recent COVID pandemic, an increasing number of approved cell and gene therapies, and other large molecule treatments, there has been increased interest in understanding the testing requirements and best methods for vaccines and biologics. Due to their unique nature, these products present new challenges for quality control. Some have very short half-lives and require rapid testing some components may enhance or inhibit certain pyrogen or endotoxin testing reagents or be inappropriate for testing in live animals. Please visit the QC Insider^® Toolbox to find useful information regarding each of these topics including: “Endotoxin Detection in Cell, Gene Therapy and Combination Products” presentation, “Endotoxin Testing for Biologics” webinar, “Endotoxin Testing for Vaccines.”

Situation in the US

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.

All biological products are required to be tested for pyrogenicity, unless the product is specifically exempted by regulation. The Code of Federal Register (CFR), Part 610, General Biological Products Standards, describes testing requirements and exemptions. Specifically: 

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.

There are exemptions for certain substances where a test method cannot be performed for release due to properties of the product (i.e., short product shelf life or toxicity of product in rabbits). Under these circumstances, a test method such as one of the Limulus Amebocyte Lysate (LAL) tests may be used as an alternative (Gel Clot LAL Assay, Turbidimetric LAL Assay, Chromogenic LAL Assay), performed as per the United States Pharmacopeia (USP) General Chapter <85> Bacterial Endotoxins Test, or an alternative test may be validated, such as a Recombinant Factor C Assay or an in vitro monocyte activation test, as described by USP Chapter <1225>. In any of these cases, prior to licensure, assurance of safety, purity, and potency must be demonstrated (21 CFR 610.9).

Situation in Europe

In Europe, RPT can only be used by exception. For assessing pyrogenicity of biologics, in vitro tests such as the MAT (Ph. Eur. 2.6.30) or BET assays (Ph. Eur. 5.1.10) including the rFC assay (Ph. Eur. 2.6.32) are recommended, after a product specific validation.

Biologic products, including vaccines and cell and gene therapies are growing modalities for disease prevention and treatment. These products present new challenges for quality control. This scheme depicting an example biologic product purification process shows some of the steps where endotoxin testing is conducted.

Additionally, raw material and final product must also be tested for the presence of bacterial endotoxins. Lonza's wide range of pyrogen and endotoxin testing solutions supports your efforts in testing, including for vaccines, cell and gene therapies and biologics. The breadth of test types, as well as scalability options, helps make your QC testing program efficient, allowing the best test method to be used for your sample types. Lonza’s expert Scientific Support Team is available to address all of your pyrogen and endotoxin testing needs.