Pyrogen and Bacterial Endotoxin Testing

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Endotoxin and Pyrogen Testing

Our comprehensive range of testing solutions supports your efforts in pyrogen and endotoxin testing of raw materials, in-process samples and manufactured product. No matter where you are in your process, Lonza’s testing products optimized with our world-class software and hardware solutions and supported by our experts will help streamline your work flows and meet regulatory requirements for injectable drugs and implantable medical devices, including for new modalities used in modern vaccines and biologics.

We can help answer your questions about endotoxin testing whether you use qualitative gel clot, quantitative turbidimetric or quantitative chromogenic LAL-based assays, or want sustainable recombinant factor C endotoxin testing or sustainable pyrogen testing.

The QC Insider^® Toolbox is a vast library of tech tips, white papers, e-learning modules and more, all in one place, all designed to help you with your pyrogen and endotoxin testing program.

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Senior Scientific Support Specialist for Lonza endotoxin testing, a troubleshooter and musician who loves the aspect of training in his daily work. Learn about Travis’ contribution to our QC Testing Solutions group, and the rewarding aspects of his job.

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In the following sections, we provide an overview of the LAL assay, including its importance in BET and how it enables the reliable and sensitive detection of bacterial endotoxins. We also outline the different methods of LAL testing that we offer, including the gel-clot test, turbidimetric and chromogenic assays, and the non-animal-derived recombinant Factor C (rFC) assay. This will help you choose the most suitable type of LAL testing method to meet the needs of your application.

What Are Pyrogens and Endotoxins?
BET and Test Types
Testing of Biologics, including Vaccines

Pyrogens are fever-inducing substances primarily derived from microorganisms such as bacteria, viruses, yeasts, molds, or chemical substances, e.g. primary packaging materials. Pyrogens entering the bloodstream may interact with the host immune system to cause inflammation, fever, chills, shock and, in severe cases, death.

Pyrogens include membrane-derived compounds like lipid-polysaccharides (LPS), flagellin, peptidoglycans, lipids, proteins, etc. They are often heat stable and therefore difficult to remove during manufacturing. The most potent type of pyrogens are bacterial endotoxins which are derived from the cell wall of gram-negative bacteria. Pyrogens that derive from microorganisms other than gram-negative bacterial are collectively referred to as non-endotoxin pyrogens (NEPs).

There are a variety of methods that can be used to detect pyrogens: The traditional qualitative approach has been the rabbit pyrogen test (RPT), which involves measuring the body temperature increase in rabbits following injection of a product potentially containing pyrogens. The monocyte activation test (MAT) was introduced as a sustainable, in vitro alternative to the use of experimental animals. The MAT is based on measuring the release of pro-inflammatory cytokines from cultured human blood monocytes in response to pyrogenic contaminants. MAT kits, such as the PyroCell^® MAT System, which measure release of IL-6 are especially useful early in drug development when the presence of all pyrogens should be examined and testing is not focused specifically on endotoxins.

Bacterial endotoxins are the most potent pyrogenic contaminants which must be excluded from all injectable drugs and implantable medical devices. The bacterial endotoxins test (BET) has widely replaced the RPT for pharmaceutical and biotechnology products. Traditional BET using Limulus amebocyte lysate (LAL) tests contain specialized blue blood cells from the wild Atlantic horseshoe crab, Limulus polyphemus, as a component because they react to the presence of endotoxins in a way that can be measured and quantitated. (Similarly, tests available in Asia include cells from an Asian horseshoe crab species, Tachypleus spp., and are called TAL assays.) Sustainable tests, using a recombinant version of the first enzyme in the LAL clotting cascade, such as the PyroGene^® rFC Assay, do not rely on the blood of horseshoe crabs. They are gaining usage across the globe as companies seek to reduce their reliance on natural resources.

The bacterial endotoxin test (BET) is a critical part of quality control (QC) testing. Testing products for the presence if 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 developed¹.

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.

Qualitative: Simple Yes/No Answer.

Gel Clot Assay:
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 are calculated from a standard curve.

Turbidimetric LAL Assay:
Kinetic measurement of turbidity development is a cost-effective way of BET testing water samples or large volume parenterals.
Chromogenic LAL Assay:
Kinetic measurement of color development allow a quantitative readout of the endotoxin concentration in the sample measured.
Recombinant Factor C Assay:
Sustainably synthesized alternative to LAL assays 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.

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 absorbance or fluorescence microplate readers. 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.

To learn more about the different endotoxin testing methodologies, access our Assay Selection and e-learning modules, featured in the QC Insider^® Toolbox, where you will find many tools to improve the efficiency of your QC testing program.

¹ Iwanaga S. (1993). The limulus clotting reaction. Current opinion in immunology, 5(1), 74–82.

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”.

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).

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.

An Introduction to Pyrogen and Bacterial Endotoxin Testing