Endotoxin Testing

Endotoxins, also known as lipopolysaccharides (LPS), are toxic substances found in the outer membrane of Gram-negative bacteria. These endotoxins are released into the environment when bacterial cells die or undergo lysis [1].

History of Endotoxin Testing

Endotoxin testing has evolved over the years, starting from pyrogen testing to more refined methods like gel clot, turbidimetric, or quantitative chromogenic Limulus Amebocyte Lysate (LAL)-based assays. These lysates are prepared from the amebocytes of the horseshoe crab, producing a clot or gel upon exposure to bacterial endotoxin [2]. Pyrogen testing, which began in the early 1900s, aimed to detect fever-inducing substances in parenteral medicines, often derived from microorganisms. The most potent pyrogens are bacterial endotoxins, which are a subset of pyrogens [3].

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Image 1: Bacterial Endotoxin Testing (BET) at Boston BioProducts. In our quality control lab, we have optimized the efficiency in which products may be tested for endotoxin, ensuring that necessary specifications are met in a time efficient manner. Here, the LAL reagent used to perform the test is loaded into our BET platform.

Importance of Endotoxin Testing

Testing for bacterial endotoxins is crucial because their presence in buffers, reagents, or solutions can lead to various experimental issues, and contaminated products can be harmful to human and living models. In humans, pyrogens entering the bloodstream may initiate secretion of pro-inflammatory cytokines by monocytes. When high concentrations of pyrogens enter the blood stream they cause fever, chills, septic shock and in severe cases even death [4]. Common sources of endotoxins in these material include the water used, raw materials, equipment, and packaging components. Controlling microbiological and endotoxin contamination in these sources is a fundamental part of good manufacturing practice.

USP <85> Bacterial Endotoxin Test

Chapter 85 of the United States Pharmacopeia (USP) sets forth the criteria for conducting Bacterial Endotoxin Testing (BET) under accepted USP standards [5]. This test includes sample suitability to ensure the interaction of horseshoe crab-derived amoebocyte lysate with endotoxins is not being interfered with, yielding a quantifiable and reproducible outcome. The use of this assay aids in the detection of detrimental bacterial endotoxins from laboratory apparatuses, and ensures their safety for downstream applications. The protocols mandated by the USP for the BET include proper system calibration, validation, and ongoing management, allowing for the constant improvement of Quality Control processes.

What test is used at Boston BioProducts?

Endotoxin testing at Boston BioProducts is compliant with USP <85> regulations, and utilizes the Sievers Eclipse Bacterial Endotoxin Testing (BET) Platform with associated Eclipse software [6]. This technology allows for a more-automated process of quantitative BET testing using a microfluidics plate. In providing horseshoe crab LAL and associated reagents to this system, endotoxin levels can be calculated and analyzed from a sample in a colorimetric measurement proportional to present endotoxin levels. With the streamlined approach and data extraction capabilities of the Eclipse platform, formulated reagents can be screened for endotoxin prior to release and shipment. 

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Images 2 & 3: Analysis of Sievers Eclipse Bacterial Endotoxin Platform data. The process involves the careful execution, use of reagents, and later examination of data. Even the slightest misstep can cause test failure.

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Image 4: The eclipse analyzer measures the time it takes for each known standard to reach the "onset time". The onset is the time at which each standard reaches the optical density threshold, or the lower detection limit of the analyzer. Using the created standard curve, the software can identify the concentration of endotoxin present in test samples based off how long each sample takes to reach the onset time.

Considerations and Limitations of Endotoxin Testing
Level of Endotoxin in Water Ensure that the water used as a solvent has an extremely low endotoxin level, and regularly monitor and maintain the water filtration system used for development of products. In manufacturing situations lacking access to such filtration systems, it is most cost efficient and safe to purchase water tested for endotoxin.
Cleanliness of the Environment and Containers Prepare buffers in a clean environment using clean containers and filter them immediately after preparation to prevent microbial contamination.
pH Adjust the pH of the solution to fall within the 6.4 to 8 pH range, as the endotoxin assay functions best in this range.
Ionic Strength High salt concentration (> 1M) can inhibit the endotoxin assay, so samples with high salt concentrations should be diluted.
Presence of Detergents Detergents can interfere with the detection of endotoxins, so buffer solutions with detergents should be diluted before testing.
Presence of Chelators Chelators like EDTA, EGTA or Citrate can inhibit the endotoxin assay, so dilution of samples with chelators is necessary.
Presence of Chaotropic Agents Chaotropic agents like urea or guanidine chloride can alter endotoxin aggregates that result in inhibition of LAL activity, so appropriate dilutions are required.
Presence of Organic Solvents High concentrations of organic solvents can dissolve lipid A, the lipid portion of endotoxins, leading to inhibition of the assay.
Presence of Sugars Impure sugars containing β-glucans can yield false-positive results, so use highly purified sugars.
Presence of Serum Proteins Serum proteins such as Bovine Serum Albumin (BSA) or Human Serum Albumin (HSA) can affect endotoxin detection, so dilute samples containing serum albumin.
Presence of DNA and Fatty Acids Nucleic acids and fatty acids can cause inaccurate results, so monitor and correct these parameters.
Physical Agents Viscosity, color, and turbidity of samples can interfere with absorbance measurements, so manipulate these parameters before testing.

In addition to Endotoxin Testing, Boston BioProducts provides a comprehensive set of QC tests for custom reagents. Learn more about custom reagent development services.

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