How To Measure Endotoxins In Medical Products Before And After Manufacturing
What is an endotoxin?
The “endo” in endotoxin refers to something that is within. The “toxin” component of endotoxin relates to something poisonous. Endotoxins can be deadly to humans and come from the cell walls of gram-negative bacteria. The endotoxins themselves are molecules with fat components (lipids) and complex sugar components (also known as polysaccharides). The fat and sugar components of endotoxins 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.
How do you measure endotoxins in medical products?
A bacterial endotoxins test (BET) is used to quantify endotoxin levels (also known as pyrogens) in a medical product. A BET provides accurate enough readings that manufacturers can calculate an endotoxin increase or reduction in materials before and after various processing steps. Endotoxin levels in raw materials, at critical control points in the manufacturing process, and in finished products can be calculated as well.
What are depyrogenation studies, and why are they important?
Depyrogenation studies are a part of endotoxin control and monitoring.
Endotoxin control and monitoring are necessary to make sure medical products are safe for patient use. Multiple variables in depyrogenation study structure and test methodology can affect the outcome of a depyrogenation study. Items to consider when designing and performing a depyrogenation study are further detailed below.
To give an overview of the steps required for a successful depyrogenation study, a testing system will include an endotoxin concentrate, an endotoxin inoculation protocol for the samples (materials, product, or equipment) to be depyrogenated, extraction or recovery methods for leftover endotoxins on the samples being tested. Also included are an appropriate BET test methodology and sensitivity test.
What do you need for a depyrogenation study?
#1: Select an endotoxin standard
Consider the source of the endotoxin to be used as the standard for the BET (purified or natural). When using purified LPS, use a preparation with no fillers. Once an endotoxin standard has been selected, use the same standard in subsequent studies to reduce variability.
#2: Determine the characteristics of the material being depyrogenated
LPS may adsorb to plastics or surfaces with special finishes or conformations. As a result, the sample materials assessed can affect LPS extraction efficiency and recovery during endotoxin testing. When it comes to solutions, formulation matrices, pH, salt concentration, chelating agents, surfactants, and divalent cations may all impact endotoxin. Selecting a natural endotoxin standard can solve some of these recovery issues.
#3: Assess the level of pre-processing activity needed
The current industry standard is to add enough endotoxin to the system to recover at least 1000 endotoxin units (EU) before depyrogenation. However, when designing a depyrogenation study for a product stream that usually contains <1 EU, a spike of 1000 EU may be excessive. Knowledge of historical levels of endotoxin exposure, the efficiency of the depyrogenation process selected, the endotoxin acceptance criteria, and the efficiency of the endotoxin recovery method are all important to setting a pre-processing activity. For studies that simulate “worst case” endotoxin exposure, it may be helpful to add in the “worst-case” level of naturally occurring endotoxin to simulate maximum endotoxin contamination for endotoxin reduction studies.
#4: The preparation of test samples
The method used to add endotoxins to the surface of materials to be depyrogenated may affect endotoxin removal or recovery. The three most common methods to introduce endotoxins to material surfaces are air-drying, freeze-drying, and vacuum drying. Air-drying is most used. Decisions for the design of depyrogenation studies must be documented and justified.
#5: Choose a recovery method
There is no standard method for the recovery of endotoxin dried onto solid surfaces that are used as indicators in depyrogenation studies. Although it is most convenient to adopt the standard extraction methods described in USP 161, a laboratory may choose to develop and validate a method that better suits the material under test. Recoveries of less than 100% of the endotoxin challenge material in positive controls are common. Thus, it is less important to get 100% recovery but consistent recovery across lots of the same material and depyrogenation studies.
#6: Select the test method
Lysate formulations for BET differ. If BET interferences such as leachables, chelators, and salts from test samples are impossible to overcome, use another BET test method (e.g., gel clot, kinetic chromogenic, kinetic turbidimetric, endpoint chromogenic) or a different lysate reagent source.
#7: Choose the depyrogenation method
Differences in the depyrogenation treatment (dry heat, chemicals, filters, etc.) may impact endotoxin recovery. Various treatment methods will work better than others and will work more effectively on some materials than others. Further, when performing depyrogenation via filtration, the type of filter chosen significantly impacts endotoxin levels post-filtration.
As a side note, the efficiency of a depyrogenation process is now measured based on measured endotoxin limits for patent safety and not on the 3-log reduction system introduced in 1984. Keep patient endotoxin limits in mind when assessing the efficiency of your depyrogenation methods and endotoxin control systems.
Summary
Overall, bacterial endotoxin tests quantify endotoxin levels and provide accurate enough readings that manufacturers can calculate an endotoxin increase or reduction in materials before and after various processing steps. Depyrogenation studies are a critical part of endotoxin control and monitoring. Endotoxin control is essential for ensuring medical devices and products are free of toxins and safe for patient use. Multiple variables in study structure and test method can affect the outcome of a depyrogenation study. Items to consider when designing and performing a depyrogenation study are further detailed above. If seeking support for endotoxin monitoring, choose a contract testing organization that can support you with appropriate 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
United States Pharmacopeial Convention. <1228> Depyrogenation. Rockville, MD, USA. 2021. (USPC <1228>).
