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What are pyrogens and endotoxins?

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 and are ubiquitous. Thus, we must try to keep the endotoxin levels as low as possible for 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. The use of an alternative method reduces the demand on a natural resource and can help meet supply chain sustainability initiatives. They are gaining usage across the globe as companies seek to reduce their reliance on natural resources.


Monocyte Activation Test

With PyroCell® Monocyte Activation Tests (MAT) we can eliminate the reliance on rabbit-based pyrogen testing, helping to ensure drug product safety and compliance. 

Learn about MAT

Run assays with the right reader

Learn about conducting traditional kinetic LAL Assays and recombinant Factor C technology in one easy-to-use instrument. Find out more about Lonza's Nebula® Readers. 

Nebula® Readers

Automation in the testing lab

Implementing the PyroTec® PRO Automated Robotic Solution can reduce the potential for human error and enhance the accuracy, reliability and traceability of results. 

PyroTec® PRO

Learn in the QC Insider® Toolbox

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

Become a QC Insider®

Topics in 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

Description Test type

Qualitative tests


Simple yes / no answer

Gel Clot Assay

This is a simple LAL test with visual inspection of gel formation. This does not require an incubating reader and software to complete.

More details

Quantitative tests


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.

More details

Chromogenic LAL Assay

Kinetic measurement of color development allows a quantitative readout of the endotoxin concentration in the sample measured.

More details

Recombinant Factor C Assay

Sustainably synthesized alternative to LAL assays based on the recombinantly produced form of Factor C. Does not rely on horseshoe crab blood.

More details

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.

Our commitment to conservation

Horseshoe crabs play a significant role in the pharmaceutical industry for endotoxin testing. Their amoebocytes, or blood cells, are a biomedical gift. The conservation of horseshoe crab populations is a fundamental priority for Lonza. Lonza is committed to protecting the horseshoe crab, adopting best practices, and actively supporting conservation efforts developed by the Ecological Research & Development Group (ERDG). ERDG is a non-profit wildlife conservation organization that focuses on conserving the four remaining horseshoe crab species.

Our annual sponsorship includes donations of products and financial support for educational programs about the horseshoe crab that help coastal communities save their breeding and nursery grounds. We do not sell the horseshoe crabs we use for biomedical purposes but return them to the waters where they are from.

Additionally, to promote the preservation of the horseshoe crab, a senior member of our staff serves on the Atlantic States Marine Fisheries Commission (ASMFC) Technical Committee for the horseshoe crab and on the joint team of the ASMFC and Shorebirds Commission.

Lonza’s Global Endotoxin Summit

Biannually at the Global Endotoxin Testing Summit, Lonza brings together testing experts, researchers, and conservationists to support ERDG’s Just Flip em® Program. This day brings a virtual event in person to a horseshoe crab sanctuary beach, where we help save horseshoe crabs stranded from spawning activities.

Global Endotoxin Testing Summit

At the Global Endotoxin Summit attendees learn from thought leaders around the world, who address topics of preservation of the horseshoe crab, current conservation efforts, adopting best practices, and more.

References

Chen J. Low Endotoxin Recovery in Common Biologics Products, presented at the 2013 Annual PDA Meeting, Orlando, FL, April 2013

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

Maggio ET. Polysorbates, peroxides, protein aggregation, and immunogenicity – a growing concern. J Excipients and Food Chemistry 2012 3:45-53

Maggio ET. Polysorbates, immunogenicity and the totality of the evidence. BioProcess International 2012 10: 44-49