It is important to adhere to current methods of endotoxin testing, on which the industry relies for process sustainability.
The potentially devastating effects of endotoxins to humans have been widely reported. At sufficient concentrations in the bloodstream it is possible for endotoxins to cause irreversible side effects and even death in severe cases.
Found on the outer cell membrane of Gram-negative organisms, bacterial endotoxins are one of the most potent toxins known and strongly pyrogenic (fever-causing) substances, explains Allen L. Burgenson, Testing Solutions, Lonza Pharma & Biotech. “These toxins are harmless outside of the body and in the alimentary canal. They are extremely potent, however, once introduced in the bloodstream or intrathecally,” he says.
“Bacterial endotoxins (also referred to as pyrogens) have the capacity to induce a high fever,” confirms John Dubczak, general manager, Microbial Solutions, Charles River Laboratories. “A large enough dose of endotoxin can lead to lung and kidney failure, intravascular coagulation, and a systemic inflammatory response that can lead to septic shock and death.”
As a result of the dangers of bacterial endotoxins, the importance of testing for them is plainly apparent, in the view of Victoria Watson, laboratory manager, Microbiology at Wickham Labs. “The importance of endotoxin testing is clear when looking at how susceptible and sensitive humans are to even minute amounts of endotoxin,” she says.
“Bacterial endotoxins are ubiquitous in nature and are a menace for the manufacturers of all parenteral drugs and medical devices,” asserts Dubczak. “Not only are they everywhere environmentally, they are very difficult to remove once they are introduced into a finished parenteral product.”
Introduction of these molecules can come from simple contact with contaminated parenteral devices or medications, notes Watson. “Ensuring that equipment and medication are free from endotoxins is particularly important when caring for vulnerable patients, including the elderly, patients in intensive care, and infants,” she says. “For example, sudden infant death syndrome has been linked to varying levels of bacterial endotoxin present in the bloodstream.”
“In the early days of injectable pharmaceutical products, there was no method for testing for pyrogenic contaminants,” reveals Burgenson. “If a patient received an injection, there was a high likelihood that they would spike a fever, which at the time was termed ‘injection fever’.”
It was the work of Florence Seibert and her team in the 1920s that brought forth the introduction of the Rabbit Pyrogen Test (RPT) (1), which was eventually incorporated into the United States Pharmacopeia (USP) in 1942 as an official test, Burgenson explains. “The clotting of Limulus blood was noted for many years in injured horseshoe crabs,” he says. “Frederick Bang and Jack Levin discovered the enzymatic pathway causing the clotting of Limulus blood (2,3), and developed the Limulus Amebocyte Lysate, or LAL test.”
During the 1970s, pharmaceutical companies experimented with the LAL test and compared it with the RPT, finding that the LAL test was a more reliable and sensitive assay (4), and offered cost-efficacy for companies as there was no need to maintain rabbit colonies for product testing. “FDA took notice, and decided that, since LAL was derived from blood, it was a biologic subject to regulation under Section 351(h) of the Public Health Service Act, and subject to regulation and licensure by FDA,” confirms Burgenson.
“Since its introduction, the LAL test has become one of the most important tools used in the pharmaceutical industry,” adds Dubczak. “It has allowed for a level of bacteria and pyrogen monitoring that simply eluded manufacturers when the RPT was the only pyrogen test available. The LAL test has ensured the absence of pyrogens in raw materials, water for injection systems, in-process samples, and in the final products.”
The LAL assay works via the reaction of the LAL reagent-an aqueous extract of blood cells from the horseshoe crab-with bacterial endotoxins and lipopolysaccharides. “There are currently three compendial bacterial endotoxin tests (BET) found in the USP (gel-clot, kinetic chromogenic, and kinetic turbidimetric) that may be used to test injectable pharmaceutical products and implantable medical devices for release into commerce,” notes Burgenson. “There is also the endpoint chromogenic assay, which is a timed endpoint version of the kinetic chromogenic assay.” (See Table I for a list of the advantages and disadvantages of endotoxin testing methods.)
Gel-clot LAL test. This method is the oldest of the three and the simplest, Dubczak explains. “The major advantage of the gel-clot LAL test is its simplicity,” he says. “It is easily learned and can be performed without expensive equipment.”
The gel-clot assay works through production of a clot in the presence of endotoxins by cleavage of a coagulogen to coagulin, notes Burgenson. “This method can be performed as either a limits test or a semi-quantitative assay, giving a positive or negative result at the sensitivity of the lysate used,” adds Watson. “Dilutions are tested with lysate, and the endpoint is used to calculate approximate endotoxin level.”
Despite the method’s advantages, such as interacting with fewer materials, thereby giving rise to a lower likelihood of results being affected by sample inhibition or enhancement, certain disadvantages have hindered its popularity. “The amount of time that is required to prepare samples using this process has made it less popular for use in raw material testing in some laboratories,” Watson says. “Additionally, the lysate sensitivities used in the gel-clot method are less sensitive than the quantitative methods.”
“Furthermore, the test does not allow for automation and data assimilation by computers, making it difficult to track and trend results,” adds Dubczak.
Kinetic turbidimetric LAL test (KTA). The KTA is one of the quantitative assays in which the measurement of absorbance versus endotoxin concentration is used to create a standard curve, explains Watson.
“During the LAL-endotoxin reaction, the solution mixture becomes increasingly turbid as the gel begins to form,” adds Dubczak. “The time required for these turbidity changes to occur is inversely proportional to the amount of endotoxin present in the sample (i.e., more endotoxin requires less time). The endotoxin in a sample can be estimated by comparing the time it takes to reach a defined level of turbidity to the times obtained from a series of endotoxin standards (standard curve).”
Sensitivity can be higher with KTA over the gel-clot method, he asserts, and although it is possible for KTA to be performed in test tubes, it is more common to perform the assays on plastic, 96-well microtiter plates. “The spectrophotometers that are used to measure turbidity changes can be captured by the use of appropriate software, and the assimilated data are used to generate quantitative reports,” Dubczak continues. “Those same data can be uploaded to a LIMS database for tracking and trending.”
Watson adds that the most significant advantages of quantitative methods over the gel-clot method are the increase in sensitivity and ability to extrapolate results. “Being able to extrapolate quantitative results can be invaluable when testing raw materials because it can offer insights into potential sources of endotoxin contamination,” she states.
Chromogenic substrate test. The third method, the chromogenic substrate test, is a further evolution of the LAL test. “Instead of cleaving coagulogen to coagulin, the same enzymes cleave a substrate-releasing para-nitroanaline (pNA), and a yellow color develops,” reveals Burgenson. “Similar to the turbidimetric assay, the time of color development to exceed a threshold is measured. The more endotoxin, the faster the substrate is cleaved, resulting in a deeper yellow color.”
For Dubczak, these chromogenic tests have an elegant methodology and feature many of the advantages that are found with KTA, such as increased sensitivity and extrapolation of quantitative results. “Chromogenic tests are, however, more expensive,” he says. “The chromogenic substrate material requires months to synthesize and as such, is very expensive to manufacture.”
“The latest evolution of the LAL assay is the use of recombinant Factor C, derived from the DNA of a horseshoe crab,” says Burgenson. “The gene sequence for Factor C is inserted into cells and expressed in cell culture. This recombinant Factor C (rFC) is the same as the natural Factor C. The assay using rFC does not produce a gel, turbidity, or a yellow color; it produces fluorescence proportional to the amount of endotoxin in a sample. The more endotoxin in a sample, the more light is generated.”
Another in-vitro alternative is the monocyte activation test (MAT), which was developed on the principle of the human immune system response, reveals Watson. “When challenged by the pyrogens entering the body or coming into contact with the bloodstream, the host’s innate immune defense mechanisms cause the monocytes/macrophages to produce prostaglandins and pro-inflammatory cytokines such as interleukin-1 (ILâ1), interleukin-6 (ILâ6) and tumor necrosis factor-α (TNFâα),” she says.
Additionally, the RPT is also an option, although this should only be considered as a final option once the LAL in-vitro methods have failed, Watson notes. “RPT is generally used for highly complex products that interfere with the in-vitro methods, and for release of products that require full pyrogen evaluation in markets that do not accept MAT as an alternative,” she explains.
There are some products, namely biological ones, that are exempt from the rabbit test as stated in US 21 Code of Federal Regulations (CFR) 610.13(b), clarifies Burgenson. Therefore, in his opinion it is prudent for the developer to look at every approach that may be able to help overcome any interferences with the LAL assay. Some formulations include a chelator (such as citrate), with a non-ionic surfactant (such as polysorbate) causing the endotoxin to be lost in the product formulation. “A magnesium chloride solution has been successfully used to overcome interferences by chelating agents such as ethylenediaminetetraacetic acid or citrate buffers. There are other proprietary products, such as Pyrosperse (Lonza) or a glucan blocker that have also been used.”
If difficulties persist, however, Burgenson recommends the best course of action is to contact the LAL reagent manufacturer, so that the work can be done in partnership. “These manufacturers have seen all types of interferences many times over, and, therefore, should be able to help,” he says.
As LAL assays are performed using an extract of blood cells from the Atlantic horseshoe crab, the prehistoric marine arthropod plays an important role in patient safety. “This animal is under legal protection,” emphasizes Watson. “Without this protection, the horseshoe crab population would be threatened.”
Dubczak concurs, highlighting the fact that reduction of animal use for pharmaceutical testing has been a well-established influencer of the industry over the years, which has seen the LAL in-vitro test replace the traditional RPT to prevent the use of hundreds of thousands of rabbits per year to test for product safety and contamination. The LAL test is an animal-derived test, meaning the animal itself is not tested upon. “The horseshoe crab’s blood is collected only to a point where the crab stops bleeding as part of a natural mechanism to protect itself,” he notes.
Additionally, there has been much work and commitment by manufacturers of the LAL reagent to conserve the horseshoe crab. “Work has included sustainability partnerships to enhance the horseshoe crab population along the mid-Atlantic shores,” says Watson. “Such projects have focused on increasing the survival rate of the horseshoe crab in early stages of life. Eggs are collected, and the crabs are grown in predation-free, controlled environments then released into their natural environment when older and stronger.”
Other projects include the “Just Flip Em” program where volunteers scan the beaches to rescue horseshoe crabs stranded on the beach on their backs, adds Burgenson. Each year many thousands of stranded crabs are rescued.
“Without the protection of the biomedical industry, these prehistoric creatures would surely become endangered, if not extinct. For this reason, it is critical that we serve as advocates for the humane treatment of these animals and strive to achieve balance between our need for this valuable material and the life of the animal that provides it,” stresses Dubczak.
“There has been an increase in raw material manufacturers requiring their products be tested for endotoxins,” notes Sophie Bell, section head for Bacterial Endotoxin and Cell Culture at Wickham Labs. “These requests come in usually due to pharmaceutical manufacturers requiring materials that do not contain detectable levels of endotoxins. The responsibility of the manufacturer is to source adequate materials for their products, so regulation of raw materials seems unnecessary; especially as different endotoxin specifications in end-products can affect the level of endotoxin contamination allowed in raw materials.”
In agreement, Dubczak highlights that product regulations are designed to set forth the standardized expectations for finished drugs, biologics, and medical devices, and as such the responsibility of sourcing adequate raw material is that of the manufacturer. Using an example from FDA guidance on pharmaceutical good manufacturing practices (5), he states that it is clear that regulators expect the sound application of science and associated risk-based rationales to be the basis for a product’s development and manufacturing process.
“For instance, manufacturers should determine the level of risk for both active and inactive raw materials, and there should be a comprehensive assessment of critical raw material formulations, ingredients of in-process buffers, and final product formulations,” he adds. “However, to place strict regulations on this process would be counter to the intent of this approach.”
Furthermore, Bell emphasizes the point that raw materials are not the only source of endotoxin contamination in end products. “Manufacturing processes can equally be a source of contamination, so the requirement for endotoxin testing in the product is still necessary. Testing of products from the beginning, middle, and end of manufacture is still advised to ensure uniformity across the manufacturing process,” she says. “Products that can be terminally depyrogenated will not require as clean raw materials compared to those that cannot.”
Despite the fact that raw materials going into pharmaceutical products are not specifically regulated, drug manufacturers use the highest grade of raw materials obtainable, emphasizes Burgenson. “Many of these materials are USP grade,” he adds. “Manufacturers will test raw materials and purified waters for the presence of endotoxins, and determine if the raw material meets its specifications and is suitable for manufacturing use.”
“What is important is that the quality of the raw materials used in production meet the standards appropriate for their intended use,” clarifies Dubczak. “Raw materials or reagents do require science-based evaluation to establish the presence or absence of deleterious endogenous or adventitious agents.”
1. F.B. Seibert, Am. J. Physiol. 71 621–651 (1925).
2. J. Levin and F.B. Bang, Bull. Johns Hopkins Hosp. 115 337–345 (1964).
3. J. Levin, P.A. Tomasulo, and R.S. Oser, J. Lab. Clin. Med. 75 903–911 (1970).
4. F. Pearson, “LAL Assay: Applications and Equivalence,” in Pyrogens: Endotoxins, LAL Testing, and Depyrogenation, Marcel Dekker, pp. 148–155 (New York and Basel, 1985).
5. FDA, “Pharmaceutical cGMPs for the 21st Century-A Risk Based Approach,” Final Report (September 2004).
Vol. 43, No. 7
When referring to this article, please cite it as F. Thomas, “Putting Endotoxins to the Test,” Pharmaceutical Technology 43 (7) 2019.