Bacterial Endotoxin Testing For Medical Devices
What is an endotoxin?
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). This is 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.
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 innate 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. The innate immune system has a single mode of action when activated by something foreign to the human body. 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 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. 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 can be performed on raw materials used to create pharmaceuticals, the final pharmaceutical formulation, and medical device products.
What is a bacterial endotoxin test (BET)?
A bacterial endotoxins test (BET) uses an assay known as the Limulus Amoebocyte Lysate (LAL) test. BET testing is considered a pyrogenicity test. However, do not confuse BET testing with rabbit pyrogen 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.
How is bacterial endotoxin testing performed?
LAL is dissolved in water or buffer solution free of endotoxins and interfering factors to create a standard endotoxin stock solution. All endotoxin stock solutions are calibrated to the current World Health Organization International Standard for Endotoxin. Endotoxin is expressed in Endotoxin Units (EU). One USP Endotoxin Unit (EU) is equal to one International Unit of endotoxin. Then a serial dilution of the endotoxin standard is prepared. Solutions for samples to be tested are prepared by immersing the device or dissolving the product in the same water or buffer solution used for the endotoxin stock solution. If necessary, the pH of the solution will be adjusted so that it falls within the range of 6.0-8.0.
A LAL gel-clot technique is used for detecting or quantifying endotoxins based on lysate reagent clotting upon endotoxin exposure. A series of tests are performed to confirm the sensitivity of the lysate solution and for any interfering factors. The sensitivity of the lysate solution is considered to be the minimum concentration of endotoxin required to cause the lysate to clot under test conditions. Both quantitative and limit testing using the LAL gel-clot technique can be performed. For brevity, only the bacterial limit test is described below.
For bacterial endotoxin limit testing of your medical device, four solutions (Solutions A, B, C, and D) are prepared. The details of these solutions are shown in Table 1 of USP 85, which has been reproduced as Table 1 below. Solutions B and C are positive controls with standard endotoxin solution at twice the concentration of the lysate sensitivity. The negative control Solution D is a negative control that consists of water or buffer solution.
A BET test is valid when all Solutions B and C samples test positive, and all Solution D samples are negative. In other words, the test is accurate when the positive controls test positive, and the negative controls test negative. If both Solution A samples test negative for bacterial endotoxin, the samples pass the BET test limits. If both Solution A samples test positive for endotoxin, the samples fail the BET test. The BET test is repeated if one of the Solution A samples tests positive and the other tests negative. Note that medical device samples do not comply with the BET testing limits if a positive test result is found for one or both Solution A sample replicates.
Special considerations for LAL testing
Amoebocyte lysate reacts to endotoxins and some β-glucans. Amoebocyte Lysates that do not respond to glucans are available. These amoebocyte lysates have the G factor reacting to glucans removed or inhibited. If you are concerned about the presence of glucans in your samples, ensure that an amoebocyte lysate is used. For medical devices, LAL testing has special considerations for product families and special medical device endotoxin limits. There are also endotoxin limits for drugs and biologics. Should your products be above their specified limits, endotoxin controls and depyrogenation studies are available to support reducing a product’s endotoxin concentration. In rare cases, traditional LAL testing may not be possible and in-vivo pyrogen testing governed by USP 151 may be needed.
Ethide Labs is a contract testing organization that specializes in Bacterial Endotoxin Testing. Ethide Labs also offers Bioburden Testing, Environmental Monitoring, Sterilization Validations, Microbiology Testing, EO Residual Testing, Package Integrity Testing & Cytotoxicity 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>).
