What Types Of Pyrogens Should You Evaluate For Your Medical Device Or Medical Product?
What are pyrogens and endotoxins?
Endotoxins come from the cell walls of gram-negative bacteria. The endotoxins themselves are molecules with both fat components and complex sugar components (also known as polysaccharides). The presence of fat components and complex sugars are why endotoxins are also known in scientific literature as lipopolysaccharides (LPS). Endotoxins are also considered pyrogens because they trigger the innate immune system and produce fever when released within the human body. Endotoxins are the most ubiquitous pyrogenic contaminants for medical devices or products. However, there are other types of pyrogens. In addition to what pyrogens are, this article will cover endogenous pyrogens, exogenous pyrogens, and pyrogen (bacterial endotoxin) testing.
Why are endotoxins harmful, and how do endotoxins cause pyogenesis?
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 natural immune system is general and non-specific. In reference to 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 LPS is 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. Endotoxin’s ability to produce a fever upon exposure in the human body is why endotoxins are called pyrogens.
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 is performed on raw materials to create pharmaceuticals, the final pharmaceutical formulation, and medical device products.
What are endogenous pyrogens and exogenous pyrogens?
Endogenous pyrogens are molecules (such as the cytokine interleukin-1) found naturally within the body and can create a fever-producing reaction when injected into (or naturally produced) by the body. Exogenous pyrogens are molecules found outside of the body, such as endotoxins from gram-negative bacteria or pyrogenic prions. Exogenous pyrogens or autoimmune diseases either provoke endogenous pyrogen production to create a fever within the body or activate a toll-like receptor (TLR) to create a fever. Clinically, the fever produced by endogenous cytokines is indistinguishable from fever produced by exogenous pyrogens such as LPS.
How do endogenous pyrogens and TLR activation relate to medical devices and products?
Cytokines are small cell-signaling proteins. Cytokines are an essential part of the immune system and are usually produced by cells when the body is ill or experiencing an allergic response. Not all cytokines are pyrogens. Some cytokines, anti-inflammatory cytokines such as interleukin-4 (IL-4), interleukin-10 (IL-10), and interleukin-13 (IL-13), reduce inflammation in the body. However, specific pro-inflammatory cytokines, such as IL-1, tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6), are true endogenous pyrogens that directly elicit the body to produce a fever. Other cytokines are by-stander endogenous pyrogens that contribute to fever production but not fever production alone. These endogenous pyrogens contrast to all bacterial endotoxins and foreign antigens, which are exogenous pyrogens. Both endogenous and exogenous pyrogens induce a fever response in the body. However, exogenous pyrogens cause fever via different means than endogenous pyrogens. Most exogenous pyrogens activate a family of cell receptors called toll-like receptors (TLRs) to create a fever. Portions of TLRs share similarities with portions of interleukin-1 (IL-1) receptors; this is of interest since IL-1 cytokines binding to IL-1 receptors on cells ultimately results in a fever response in the body. Thus, TLRs signal via the same pathways within our cells as do IL-1 receptors; this means that the body can increase body temperature in response to infection with multiple cellular pathways. It is vital to keep both fever-responsive pathways in mind when designing novel drugs or combination therapeutics for patients.
As mentioned above, exogenous pyrogens (such as LPS and other microbial agents) can trigger TLRs and induce fever. TLRs also produce pyrogenic cytokines in disease states. In fact, during infection, TLR likely directly elicits a fever response in the body and produces cytokines that further increase fever production in the body. Thus, exogenous pyrogens such as LPS can directly trigger a fever response without endogenous pyrogens through TLR activation and can further trigger a fever response in the body through TLR-mediated production of endogenous pyrogens. When it comes to medical devices and products, only exogenous pyrogens, not endogenous pyrogens, need to be evaluated. However, it is crucial to understand how a fever is triggered in the human body to avoid triggering TLR signaling pathways and pro-inflammatory cytokine production by means outside of bacterial endotoxins, such as the chemistry of various materials or drugs.
What is a bacterial endotoxins test?
A bacterial endotoxins test (BET) uses an assay known as the Limulus Amoebocyte Lysate (LAL) test. BET testing is an in-vitro pyrogenicity test and the most common test for pyrogenicity of medical devices and products. BET testing is separate from rabbit pyrogen testing. The BET rabbit pyrogen testing 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 as to the concentration of endotoxins in a sample.
Summary
Overall, pyrogens can be endogenous (produced from within the body) or exogenous (from outside). Endogenous pyrogens are molecules (such as the cytokine interleukin-1) found naturally within the body and can 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. Exogenous pyrogens either provoke endogenous pyrogen production to create a fever within the body or activate toll-like receptors (TLRs) to create a fever. Clinically, the fever produced by endogenous cytokines is indistinguishable from the fever produced by exogenous pyrogens such as LPS. Both exogenous and endogenous pyrogens are important to consider when developing a medical product so that patients do not experience a pyrogenic reaction when the product is in use. When outsourcing regulatory testing, ensure you choose a contract testing organization that can support you with appropriate pyrogen testing for your unique medical device or product needs.
Ethide Labs is a contract testing organization that specializes in Pyrogen Testing, like Bacterial Endotoxin Testing. Ethide Labs also offers Package Integrity Testing, Bioburden Testing, Environmental Monitoring, Microbiology Testing, Sterilization Validations & Cytotoxicity Testing, EO Residual Testing services for medical device companies and allied industries. Ethide is an ISO 13485 certified facility.
References
United States Pharmacopeial Convention. <85> Bacterial Endotoxins Test. Rockville, MD, USA. 2021. (USPC <85>).
United States Pharmacopeial Convention. <151> Pyrogen Test. Rockville, MD, USA. 2021. (USPC <151>).
