Bacterial endotoxin is a toxic biologic compound of significant medical importance. It is most often broadly characterized as a pyrogen, which describes a substance that acts to elicit fever in a susceptible host. Pyrogens are classified into two groups: microbial-based (sourced from bacteria, fungi, viruses, and their products) and non-microbial-based (eg, drugs, steroids, plasma fractions, or other chemicals). The most significant microbial pyrogens have been found to be endotoxins from gram-negative bacteria. Although gram-positive bacteria, fungi, and viruses can be pyrogenic during infection stages, they do so through a different mechanism and to a lesser degree than gram-negative bacterial endotoxins. Whereas bacterial endotoxins are pyrogenic, the pathologic effects caused by these substances are much more profound than just fever. These effects can include meningitis and a rapid loss of blood pressure such as in the highly and rapidly lethal condition known as septic shock that sometimes occurs in infections with gram-negative bacteria caused by the introduction of bacterial endotoxin into the circulatory system. Endotoxin may also lead to other initially latent effects as immune stimulators when introduced in the body that is the subject of further research.1
Bacterial endotoxin is a component of the outer membrane of gram-negative bacteria (Figure 66.1
Chemically, bacterial endotoxin is a high-molecularweight lipopolysaccharide (LPS) typically consisting of three distinct regions (see Figure 66.1
). The outermost region is the O-antigen or O-polysaccharide that extends outward from the cell and is attached to a central polysaccharide core. The innermost region, also attached to the core but extending into the cell wall, is a compound called lipid A, which is a disaccharide of glucosamine highly substituted with amide-linked and ester-linked long-chain fatty acids. Lipid A is responsible for most of the biologic reactivity associated with endotoxin.
Bacterial endotoxins are constantly released from gram-negative bacteria into the environment as a result of cell division, damage, or lysis and can pose a biologic threat apart from the intact microorganism if introduced to a susceptible host in sufficient quantity. Extracellular bacterial endotoxin in nature is usually associated with other outer membrane components such as proteins or phospholipids. Endotoxin contamination is difficult to prevent because it is prevalent in the environment, it’s chemical stability makes it quite challenging to inactivate, and it’s size allows it to easily pass through conventional microbially retentive filters. Due to its chemical stability, bacterial endotoxins are generally not considered to be significantly affected by many sterilization methodologies (with the notable exception of dry heat; see chapter 28
) and can therefore persist in or on various medical products or surfaces after they have been rendered “sterile.” Thus, bacterial endotoxin represents a clinical hazard if present in sufficient quantity and is introduced into certain areas of the body, particularly the vasculature, the lymphatics, the central nervous system, or the intraocular space. For these reasons, bacterial endotoxin contamination is a primary concern for manufacturers of parenteral pharmaceuticals and certain medical devices that are used to access susceptible anatomical sites.
Conversely, bacterial endotoxin is prevalent in the alimentary canal; therefore, oral dosage forms and medical devices intended for indications in the gastrointestinal tract do not generally pose the same risk of pyrogenicity.
CONTROL OF BACTERIAL ENDOTOXINS
Endotoxin, or LPS, is located in the outer layer of the dual-layered cell wall separated by a thin layer of peptidoglycan that protects gram-negative bacteria from their environment (see Figure 66.1
). Because LPS is located on the outer membrane, it possesses several properties that tends to activate a number of host defense mechanisms.
A single gram-negative bacterial cell wall is estimated to contain approximately 3.5 million LPS molecules occupying about 75% of the cell’s outer surface area.4
The LPS molecules provide several vital functions for the microorganism including structural, physiologic, and transport operations. No gram-negative bacterial species have been found to be able to survive when totally lacking LPS.5
Bacterial LPS molecules are shed from the cell into the environment through various processes such as multiplication, death, and lysis as well as constant sloughing as outer membrane vesicles (OMV) in a manner analogous to shedding of hair or skin in animals. As these processes take place, the concentration of endotoxin can accumulate and attach to dust particles, raw materials, and/or components that they come into contact with. It is important to note that bacterial endotoxin can come from the direct presence of intact microorganisms or from released, extracellular endotoxin (eg, in water or dust particles) as the source of contamination for health care products. Because gram-negative bacteria are found in virtually every environment on earth, bacterial endotoxin is also equally common in the environment. Given its ubiquity, it is interesting to speculate why endotoxin elicits such an acute response in multicellular animals to its presence in the blood stream. This response is likely due to a type of “early warning system” related to the pathogenicity of many gram-negative species.6
In fact, a study of bacterial infection in horseshoe crabs led to the discovery of a blood component in this organism that is used as a reagent (Limulus
amebocyte lysate [LAL]) for the quantitation of bacterial endotoxin. Another reason that significant levels of bacterial endotoxin are often found separate from significant levels of viable microorganisms is the stability of the LPS molecule. Bacterial endotoxin is extremely heat stable and remains active after typical moist heat sterilization and desiccation. They can pass through sterilizing filters that remove whole bacterial cells from liquids such as parenteral solutions. The hydrophobic lipid ends of the molecules are believed to facilitate the adherence to hydrophobic surfaces such as glass or plastic, which are used for many medical devices and container closures for pharmaceutical products.
A representation of the cell wall of gram-negative bacteria (left) and the outer membrane lipopolysaccharide molecule (right). Panel A reprinted with permission from Aschenbrenner and Venable.3 Figure 39-1
The production of pharmaceuticals and medical devices, particularly those with a use profile that would require nonpyrogenicity, almost always takes place in a microbiologically controlled environment. Manufacturing operations must be designed to minimize the presence of bacterial endotoxin on the product. They should operate within a state of control and assess for factors that could contribute to the presence of endotoxins (with examples given in Table 66.1
). From these examples, it is obvious that processes that expose products to water or other aqueous solutions are generally the highest risk as a source of endotoxins. In fact, dry products that are produced in a controlled environment normally have a significantly lower risk of endotoxin, as do manufacturing steps in which water is not used in the process. Manufacturing processes that involve the use of water (particularly when used at ambient temperatures) are at a higher risk because many vegetative microorganisms typically reproduce in aqueous environments, and some gram-negative bacteria are particularly adept at growing even in purified water with very little available nutrients. Therefore, it is important when seeking to control endotoxins to give primary consideration to exposure to an aqueous environment in all phases of the manufacturing process, including at raw material and other suppliers. Also, natural materials can be a significant source of bacterial endotoxin such as animal or tissue-based products.
TABLE 66.1 Examples of common sources of endotoxins in manufacturing operations
Supplied raw materials or components
Aqueous washing or finishing processes
Drying or curing processes
Aqueous leaching or soaking
Product or material storage
Containers used for transportation or storage during movement between manufacturing areas that are not appropriately cleaned
Product stored in a humid environment that supports the proliferation of microorganisms
Overall, manufacturing processes should be designed and controlled to reduce the risk of potential sources of endotoxins. Considerations for these control measures may include:
Supplier quality such as monitoring endotoxin level of incoming raw materials, components, and subassemblies. This also might include supplier certification or attesting to no contact of product with water.
Control and monitoring of process water or other process solutions. Control may be demonstrated by microbiologic testing, endotoxin monitoring, or monitoring of control measures such as temperature or chemical disinfectants/preservatives such as chlorine.
Monitoring of in-process product at specified control points. This could include product endotoxin testing or manufacturing control parameters such as time or temperature.
Regularly scheduled cleaning, disinfection, and maintenance of equipment, particularly those that convey or contain product or aqueous processing materials
Microbiologic control of the environment and associated processing materials
Another control measure that might be used to eliminate endotoxins is through depyrogenation of materials, equipment, and/or final finished products. Depyrogenation can be accomplished by two primary means: inactivation or removal. Inactivation is accomplished by denaturing or destroying the LPS molecule by chemical or physical means. Due to the inherent chemical stability of LPS, the extent of treatment necessary to achieve inactivation of endotoxin is often severe and often beyond the capability of most health care product to withstand. Chemical inactivation involves treatment to break chemical bonds or bind active sites. Physical inactivation typically involves incineration or dry heat inactivation of LPS at high temperatures. Endotoxin removal can be achieved by several techniques related to the physical characteristics of endotoxin such as size, binding affinity, solubility, and molecular weight. Where possible, manufacturing processes may incorporate design features that can act to mitigate the potential for endotoxins. Some examples of these might include the following:
Exposure to dry heat: typically, temperatures greater than 250°C for adequate duration
Acid or base hydrolysis
Distillation, ultrafiltration, reverse osmosis
For chemical and physical depyrogenation, it is often very difficult to achieve parameters that provide the necessary effectivity while preserving product activity and/or function. Depyrogenation by dry heat is the most often used method and is widely used in laboratory and manufacturing operations for treatment of heat-tolerant
items such as glassware, metal equipment, and some heat-stable chemicals. Depyrogenation by this method is typically accomplished by exposure to temperatures of not less than 250°C for not less than 30 minutes.7
Validation of such processes is typically required to demonstrate their effectivity.
NONPYROGENIC HEALTH CARE PRODUCTS
Many health care products, both pharmaceuticals and medical devices, are required to be nonpyrogenic. Sterile parenteral drugs and implantable medical devices that contact nonintact tissue during use or that have indirect intravascular, intralymphatic, intrathecal, and intraocular contact are recommended to be nonpyrogenic. The claim of “nonpyrogenic” refers to endotoxin derived from gram-negative bacteria.8
This requirement can be related to a label claim of “nonpyrogenic,” the intended use of the product, or both. The substantiation of a product as being nonpyrogenic is made by demonstrating that the product does not expose the individual on which the product is used to levels of bacterial endotoxin above specific regulatory limits. These limits can vary depending on the product and its intended use. The general regulatory limits for these products are listed in Table 66.2
Regulatory limits for bacterial endotoxina
Regulatory Limit (EU = Endotoxin Unitb)
Parenteral drug—administered per surface area
aFrom International Organization for Standardization,9 US Food and Drug Administration,10 American National Standards Institute,11 US Pharmacopeial Convention,12 US Food and Drug Administration,13,14 and US Pharmacopeial Convention.15,16
b Endotoxins are measured in EU. This is a standard unit of measure for endotoxin activity established relative to the activity contained in 0.2 ng of the US Reference Standard Endotoxin Lot EC-2 (US Pharmacopeia standard reference material).
Endotoxin limits for devices are considered more straight forward as they are generally considered as conservative with a defined endotoxin content per device, no matter the size or intended use, although even more conservative levels as defined for devices for intrathecal or intraocular use.9
But the label claim for devices can vary such as “nonpyrogenic fluid path” in which only the surfaces relevant to the fluid path have patient contact and would therefore only be considered a risk for those parts of the device. Also, devices can contain components or regions that do not have patient contact and are inappropriate to include in product testing such as a device-associated handle or cord. The exemption of any part of a product from evaluation for a “nonpyrogenic” claim should be based on an evaluation of the patient contact profile of the product. Some multiple component device products such as kits or trays can contain components that have applicable patient contact and others that do not. Typically, the “nonpyrogenic” claim only applies to those devices or components that have applicable patient contact.
Pharmaceutical endotoxin limits are also managed based on the patient contact or dosage form; however, the limits are typically based on the product dose administered. The limits for pharmaceuticals are typically expressed as K/M, which is a dose concentration-based approach where
is the regulatory limit as described in Table 66.2
M represents the maximum human dose per kilogram that would be administered in a single bolus or continuously over a single 1-hour period.
For example, the calculation of the endotoxin limit for a nonintrathecal drug administered parenterally at 2 mL/person would be
Maximum dose per kg assuming 70 kg
body mass = 2 mL/70 kg = 0.0286 mL/kg
Endotoxin limit (K/M) = [5.0 EU/kg] / [0.0286 mL/kg] = 175 EU/mL
But the endotoxin limit for a dose of 2 mL/person administered intrathecally would be
Maximum dose = 0.0286 mL/kg (see above)
Endotoxin limit (K/M) = [0.2 EU/kg] / [0.0286 mL/kg] = 7 EU/mL
And a topical drug administered based on a defined surface area with a maximum recommended dose of 30 mg/m3 would be
Endotoxin limit (K/M) = [100 EU/m3] / [30 mg/m3] = 3.33 EU/mg
The limits for radiopharmaceuticals are typically expressed as EU/V where
is the regulatory limit as described in Table 66.2
V is the maximum dose in milliliter. This is a whole-body dose not dose per kilogram.
Therefore, for a radiopharmaceutical with a maximum dose of 7 mL, the endotoxin limit would be 175 EU/7.0 mL, which is 25 EU/mL.
Application of any nonpyrogenic label claim or statement to health care products must be substantiated. This substantiation can be verified by directly testing each finished batch of a product to quantify the endotoxin content. For medical devices, options other than testing each finished batch are considered acceptable, and these options are discussed later in this chapter. The term pyrogen free has been historically used in some instances as a label claim; however, such a claim is generally inappropriate because it implies a complete absence of bacterial endotoxin and this cannot be demonstrated empirically due to the detection limits inherent to current test methods.