Endotoxin Testing of Buffers and Reagents

Medical and pharmacological research and development require reagents and buffers that are free of endotoxin contamination. The presence of endotoxins (nonviable bacterial components), causes issues of varying severity, depending on the use of the reagent or buffer. In laboratory research, problems range from variable results in cell culture experiments to severe immune responses in animal models, potentially resulting in death. In the production or clinical trial phases of therapeutic development, the consequences of endotoxin contamination are more serious with the risk of patient exposure leading to fever, septic shock, and even death.

Endotoxin testing is required to eliminate issues associated with using contaminated reagents. The endotoxin test is distinct from the sterility tests that detect live microorganisms. This article introduces endotoxins and explains what endotoxin testing is, why it is important, and how endotoxin testing at Teknova is performed to ensure product quality.

What are Endotoxins?

Endotoxins are the most common pyrogens, defined as substances that can induce fever. Specifically, they are the lipid A component from the outer membrane of the cell wall of Gram-negative bacteria that is typically released upon cell death or lysis. In addition to increasing variability in cell culture experiments, endotoxins can cause mild to severe immune responses in animal models and humans. Severe reactions can lead to death. Therefore, sterility testing (the detection of live microorganisms) alone is not enough to ensure the high quality of products necessary for research and pharmaceutical development. The significant burden posed by endotoxins requires reagent and buffer manufacturers to specify the endotoxin content of these products after conducting endotoxin testing.

What is Endotoxin Testing?

Endotoxin testing is the process by which manufacturers detect and quantify the endotoxins in reagents for laboratory and clinical use. Knowing the exact levels of endotoxins in buffers and reagents provides additional confidence in these products.

The United States Pharmacopeia (USP) specifies how buffer and reagent manufacturers are to conduct these tests. The bacterial endotoxins test (BET) is described in USP Chapter 85 (USP <85>). The BET is implemented at Teknova at every step of the manufacturing process, resulting in high-quality products that have been rigorously inspected for endotoxins.

Method of Endotoxin Testing at Teknova

The most common method of endotoxin detection is the Limulus amebocyte lysate (LAL) test. LAL is extracted from the amebocytes (immune-responsive blood cells) of the horseshoe crab, Limulus polyphemus. When the amebocytes detect endotoxins (a sign to the immune system of an invading pathogen), the proteins involved in the immune response clot around the endotoxins. This reaction forms the basis of the LAL test, which involves a series of enzymatic steps, and is used in different methods to test for endotoxins. The LAL test is well established, having originated in the 1970s and is the required choice for compliance with USP <85>.

The most commonly employed methods for the LAL test are gel-clot, turbidimetric, and chromogenic detection (see Table 1). We use the chromogenic method, which combines high-throughput detection with high sensitivity.

In the chromogenic LAL assay, the sequence of enzymatic reactions from endotoxin exposure results in the release of a yellow substrate that can be measured by spectrophotometry. This substrate can be detected at the reaction endpoint or monitored kinetically over time. At Teknova, we employ the kinetic method, which is significantly more sensitive than the endpoint method. The limits of detection are 0.005 EU/mL for the kinetic and 0.1 EU/mL for the endpoint method, where EU is one International Unit of endotoxin.

Table 1. Comparison of Limulus amebocyte lysate (LAL) test methods.

LAL test method What is measured? Qualitative or quantitative Limit of detection (EU/mL)*
Gel-clot Presence or absence of a clot Qualitative/Semi-quantitative 0.015
Turbidimetric Turbidity Quantitative 0.010
Chromogenic Yellow color Quantitative 0.005

* The limit of detection is the lowest amount of endotoxin that can be measured.

The presence of certain substances can lead to test interference, which manifests as either test enhancement or inhibition. However, including a positive product control (the product with endotoxin added) helps alleviate these challenges. We adhere strictly to the USP guidelines, which require this control for every sample. Additionally, we employ different methods to manage endotoxin masking, also known as low endotoxin recovery. Options include adding diluents to the reaction wells to increase endotoxin recovery and using additives to reduce the likelihood of interference.

To ensure product quality, we measure endotoxin levels at every step in the manufacturing process: water for production, prefiltration, and final product. The standard limit at Teknova is <0.5 EU/mL. However, this value can be customer-defined to meet the needs of a particular application.