Pyrogens Vs. Bacterial Endotoxins For Regulatory Testing
What is an endotoxin?
The “endo” in endotoxin refers to something that is within. The “toxin” component of endotoxin relates to something poisonous. When we combine these definitions, endotoxins are poisons from within. Indeed, endotoxins are deadly to humans because they trigger the innate immune system and produce fever when released within the human body. Endotoxins come from the cell walls of gram-negative bacteria. The endotoxins themselves are molecules with both a fat component and a complex sugar component. Complex sugars are also known as polysaccharides. The presence of fat and sugar components is why endotoxins are also known in scientific literature as lipopolysaccharides (LPS). Endotoxins are the most ubiquitous pyrogenic contaminants for medical devices or products. However, there are other types of pyrogens.
What are pyrogens?
Pyrogens are any molecules or substances that cause a feverous reaction when they enter the human body. Endogenous pyrogens (such as the cytokine interleukin-1) are found naturally within the human body. Endogenous pyrogens create a fever-producing reaction when naturally produced by the body. Exogenous pyrogens are molecules found outside of the body, such as endotoxins from gram-negative bacteria or pyrogenic prions. For context, prions are misfolded proteins that can transmit their misfolded shape onto normal versions of the same protein. Prions are of most concern for medical products and devices that interact with brain or spinal tissues. Exogenous pyrogens either provoke endogenous pyrogen production to create a fever within the body or activate the body’s toll-like receptors (TLRs) to develop a fever. Clinically, the fever produced by endogenous cytokines is indistinguishable from fever produced by exogenous pyrogens such as LPS. For additional information on pyrogens, please read our other article on endogenous and exogenous pyrogens.
Why are endotoxins harmful, and how do endotoxins cause pyrogenesis?
The lipopolysaccharide components that endotoxins are composed of are foreign to the human body. Thus, our immune system identifies LPS as something foreign that it should get rid of quickly. Compare our body to a house and our immune system to the inhabitants of a home. In this situation, the innate immune system treats LPS like an uninvited guest (such as a burglar or a rodent). The innate immune system entered does its best to get the uninvited guest (LPS) out of the house (the human body).
Though the immune system is commonly referred to as a single entity, there are two types of immunity, our innate immune system, and our adaptive immune system. The innate immune system is the immune system we have at birth. The innate immune system is general and non-specific. In the example above, the innate immune system can identify an uninvited guest but cannot determine that the uninvited guest is a rodent or a wasp. When activated by something foreign to the human body, the innate immune system has a single mode of action.
The immune system will raise the body temperature and release signaling proteins called chemokines and cytokines to increase circulation and recruit immune cells to the site of infection. This results in the pain, warmth, swelling, and redness experienced when you cut your finger or the fever and chills you experience when you have the flu. The adaptive immune system is specific, and we develop it over time. The adaptive immune system is what we activate when we vaccinate our body against specific bacterial or viral strains.
When LPSs are detected within the human body, the innate immune system is activated. The activation of the innate immune system by these endotoxins results in a fever (pyrogenesis). If endotoxins run unchecked within the human body, an individual can become septic and die. Thus, appropriate bacterial endotoxin testing for medical devices and products is vitally important to patient safety. Endotoxin testing can be performed on raw materials used to create pharmaceuticals, the final pharmaceutical formulation, and medical device products.
How do pyrogens, endotoxins, and microbes relate?
Endotoxins are the most ubiquitous source of pyrogens for medical products and medical devices. Endotoxins come from the cell wall of microbes, specifically the cell wall of gram-negative bacteria. The endotoxins are not gram-negative bacteria themselves. However, the concentration of gram-negative microbes in a manufacturing environment impacts the endotoxin concentration of a manufactured medical product or device. Thus, the better an environment reduces microbial burden (particularly gram-negative microbial burden), the better protected a manufactured medical device or product is from high levels of endotoxin contamination.
What is a bacterial endotoxin test?
A bacterial endotoxin test (BET) uses an assay known as the Limulus Amoebocyte Lysate (LAL) test. BET testing is an in-vitro pyrogenicity test. BETs are the most common, easiest, and cheapest tests of product pyrogenicity. BET testing is separate from rabbit pyrogen testing, which is rarely needed for regulatory testing. LAL is an extract of blood cells from the Atlantic horseshoe crab. LAL detects the LPS of the cell wall of gram-negative bacteria, even if these bacteria are dead. LAL detects LPS through clotting and gelling in the presence of LPS, allowing for precise calculations to be made as to the concentration of endotoxins in a sample.
LAL testing has special endotoxin limits for medical devices and special considerations for medical device product families. There are also special endotoxin limits for drugs and biologics. Should your products be above their specified limits, endotoxin controls and depyrogenation studies are available to reduce a product’s endotoxin concentration. In rare cases, traditional LAL BETs may not be possible, and in-vivo pyrogen testing governed by USP 151 may be needed.
Why is bacterial endotoxin testing important?
Bacterial endotoxin testing is an important quality control step that detects the level of endotoxin contamination of a product at any stage, from initial product manufacture to final distribution. As microorganisms exist on every surface (including our body), bacterial endotoxins can be accidentally introduced during the manufacturing or packaging process in many ways. Some of the most common examples are contamination through the raw materials used, technicians, tubing/piping used to transfer product between development stages in a process, or the manufacturing environment itself. With such abundant sources of contamination, regular endotoxin testing supports the long-term control of manufacturing sites. Bacterial endotoxin testing is primarily for medical products or devices that will be inserted into the human body. Suppose bacterial endotoxins exist on the surface of a medical device or product or are present within an injectable product. In that case, the innate immune system of the patient exposed will be activated, causing fever, chills, and potentially death.
Summary
Overall, testing for pyrogens, particularly bacterial endotoxins, is an imperative safety metric for regulatory approval of a medical device or product. Bacterial endotoxin testing ensures that medical devices and products are free of pyrogenic endotoxins so that patients will not be at risk of pyogenesis or infection following device exposure or product use. Ensure you choose a contract testing organization that can support you with appropriate bacterial endotoxin testing for your unique medical device or product needs.
Ethide Labs is a contract testing organization that specializing in Bacterial Endotoxin Testing. Ethide Labs also offers Microbiology Testing, Sterilization Validations, Bioburden Testing, Package Integrity Testing, Cytotoxicity Testing, Environmental Monitoring and EO Residual Testing services for medical device companies and allied industries. Ethide is an ISO 13485 certified facility.
References
Charles A. Dinarello. Review: Infection, fever, and exogenous and endogenous pyrogens: some concepts have changed. Innate Immunity. August 1, 2004.
Galanos C. and Freudenberg M. A. Bacterial endotoxins: biological properties and mechanisms of action. Mediators of Inflammation. 1993; 2(7): S11–S16.
United States Pharmacopeial Convention. <85> Bacterial Endotoxins Test. Rockville, MD, USA. 2021. (USPC <85>).
