Bacterial Endotoxin Testing, Part 1: Overview
By Yadnyesh Patel, microbiology subject matter expert
Due to the possibility of serious illness or death, all injectable pharmaceutical products and implantable medical devices that come into contact with the bloodstream or spinal fluid are tested for endotoxins to ensure patient safety. These products and devices are tested for endotoxins using bacterial endotoxin testing (BET) to ensure that sterile pharmaceutical products are safe for human use.
What Are Endotoxins?
Endotoxins are lipopolysaccharides, agents of pathogenicity of Gram-negative bacteria implicated in the development of Gram-negative shock.
Endotoxins are found in the cell walls of Gram-negative bacteria, which can induce inflammation and fever as an immune response in higher organisms. Reaction to endotoxins can lead to anaphylactic shock and death of patients. Endotoxins are bacterial structural components that are released when such a cell is lysed. These components are toxic if administered to humans and/or animals, causing a pyrogenic response (rise in body temperature). For this reason, it is important that drugs and medical devices that are either injected or implanted must be tested for their endotoxin content. With regard to medical devices, those with direct or indirect contact with the cardiovascular system, lymphatic system, or cerebrospinal fluid should undergo BET.
What Is The Difference Between Pyrogens And Endotoxins?
Pyrogens are fever-causing agents; they are the agents that induce the rise in body temperature in mammals, and they are possible contaminants in parenteral products. Many substances can cause fevers when injected, infused, implanted, or when they come in contact with the bloodstream or cerebrospinal fluid of mammals. Some biologics, vaccines, and cell and gene therapies may, by their nature, elicit pyrogenic responses in patients.
The predominant and most potent pyrogenic contaminants in the manufacturing of parenteral drugs and medical devices are bacterial endotoxins, which are components of the cell walls of Gram-negative bacteria (GNB).
But remember, all pyrogens are not endotoxins, but all endotoxins are pyrogens.
Endotoxins are integral with the outer cell membrane of GNB. If GNB cannot grow, endotoxins cannot be generated. However, endotoxins may remain active in cell wall fragments after cells die, so a material may be sterile but may still contain quantifiable levels of endotoxin activity. Bacterial endotoxins, when present in parenteral products (including biological products) or medical devices, indicate that the growth of GNB occurred at some point during manufacture of the product. Endotoxins can be introduced into the process stream by pharmaceutical ingredients including water, raw materials (particularly from natural sources), the active pharmaceutical ingredients (API), drug product formulation excipients, primary (product contacting) packaging components, improperly cleaned or stored manufacturing equipment, and/or ineffective microbial contamination control practices.
How Can Endotoxins Be Detected In Drug Products/Medical Devices?
There are several methods available for conducting the endotoxin test, including the in vivo rabbit pyrogen test and several in vitro alternatives that utilize the Limulus amebocyte lysate (LAL) system.
The rabbit pyrogen test (RPT) was initially adapted by the pharmaceutical and food industries, as well as used in research experiments. However, the disadvantages of the RPT for determining bacterial endotoxins are numerous: It is a long test and, therefore, the temperature of the animal needs to be measured during the 3 hours following the injection, at approximately 30-minute intervals.
When the goal is to check whether endotoxins exist in a sample, the LAL method has largely replaced the rabbit pyrogen test. As we will see in this article, there are various reasons why the LAL method is preferable to the rabbit test.
Bacterial endotoxin testing can be accomplished by the following three LAL methods:
1. gel clot
2. kinetic chromogenic
3. kinetic turbidimetric assays.
The LAL methodology is also used for the evaluation of medical devices such as single-use disposable equipment and implants. This is done by extracting the test product with pyrogen-free water (PFW) and testing for the presence of endotoxin in the extracts.
The gel clot was the original LAL method and relies upon the operator to distinguish the formation of the gel clot in the reaction tubes. It is a qualitative or semi-quantitative test that is used to screen for the presence of endotoxins. A clot formation is interpreted as a positive result for the presence of endotoxin, and if no clot forms, this is interpreted as the sample being endotoxin free. The results are from the subjective interpretation of the clot formation.
Certain cell-wall lipopolysaccharides (i.e., endotoxins) of this bacterial group lead to gelation of blood cell (amoebocytes) lysates of the Limulus polyphemus crab. It is most often mentioned as the official referee test in pharmacopeial monographs. The gel clot LAL assay involves mixing the LAL reagent with the test sample in a tube, which is then incubated. If endotoxin is present in the sample, the clotting response by the LAL reagent will be initiated.
How does the LAL test work? A drug sample is incubated within the LAL test reagent. If the drug sample is contaminated with endotoxins, a process initiates within the horseshoe crab amebocytes involving the chained activation of a cascade of enzymes, which culminates in clotting. In the case of classical LAL, the clotting that is observed signals endotoxin contamination.
Is The LAL Test Approved By Regulators?
The United States Pharmacopoeia (<85> and <1085>) and European Pharmacopoeia (2.6.32) approve the LAL test as a compendial assay for endotoxin detection. As the LAL test does not constitute a pyrogen test, the pharmacopoeia mandates the test as release parameter for parenteral drug substances.
The gel clot LAL test method requires no detection equipment or software, simply the human eye to determine whether gel clotting has taken place after drug incubation. This purely qualitative LAL test method is simple, economical but low on sensitivity (LoD of 0.015 to 0.5 endotoxin unit/ml); it is adequate for swift and informal testing during the initial stages of a product's development.
Even though RPT has a much lower sensitivity than LAL, RPT does test for both endotoxins and non-endotoxin pyrogens (NEPs) as a qualitative test. The Limulus lysate test can only test for endotoxins, but it is very capable of doing so. That’s why in 1977 the FDA allowed LAL as a replacement of RPT for detecting endotoxins in pharmaceutical and medical devices. All international pharmacopeias recommend LAL as the method to test for endotoxins. Even so, it is still a requirement in the U.S. to test a new drug for NEPs at least once. Many pharmacopeias also recommend, wherever possible, replacing tests that make use of animals with tests that don’t, and both RPT and LAL make use of animals for their tests. LAL, however, takes the blood from the horseshoe crab and uses it outside of the creature’s living body, whereas with RPT the drugs that need to be tested are injected into the rabbits.
In my next article, I’ll discuss prerequisites and reagent preparation with regard to conducting a gel clot LAL test.
About The Author:
Yadnyesh Patel earned his master’s degree in microbiology from KBCNM University, Maharashtra, India, in 2009. With over 13 years of extensive experience in quality functions, he has spent time working at pharmaceutical organizations such as Claris Otsuka Ltd, Sun Pharmaceutical Industries Ltd., and Zydus Life Sciences Ltd. Currently, at Zydus group, he spearheads microbiological quality functions, document management, audit, and compliance. He possesses substantial expertise in quality management systems, SOPs, documentation management, microbiological test method validations, sterility assurance, aseptic process simulation, and computer system validation.