Time to stop horsing around?
14 Jul 2015 by Evoluted New Media
A threat to data quality and even a real danger to human health; it is vitally important to understand the effects of endotoxins
A threat to data quality and even a real danger to human health; it is vitally important to understand the effects of endotoxins. But is it time to change the way we detect them?
Endotoxins are lipopolysaccharide complexes associated with the outer membrane of Gram-negative bacteria, which can elicit a number of undesirable pyrogenic effects in humans and animals, including septic shock, fever, vascular collapse and even death. Pharmaceuticals such as vaccines, implantable medical devices, and intravenous drugs and fluids can become contaminated with endotoxins leading to serious consequences. It is therefore of paramount importance that thorough screening of medical products and pharmaceuticals is conducted prior to patient administration.
Endotoxins can also contaminate in vitro cell cultures, such as those used in research and bioproduction, impacting cell growth and function.It is vital that researchers safeguard against endotoxin contamination.
Initially, endotoxins were detected by injecting test samples into rabbits and monitoring them to determine whether they developed a fever, which is indicative of a positive result. This practice was far from ideal, for a number of obvious reasons. Fortunately, in the 1950’s, Frederick Bang and Jack Levin discovered that endotoxins provoke the intravascular clotting of horseshoe crab blood (Limulus polyphemus)1,2,3. The careful use of blood lysates from the animal was later approved for commercial applications, and the Limulus Amoebocyte Lysate (LAL) assay has since grown to accommodate the needs of both industry and research scientists. These applications cover a broad spectrum of assay sensitivity requirements, turnaround times, frequency of use, and equipment availability. This has resulted in the development of a number of methods utilising the technique, including gel clot, endpoint chromogenic, kinetic turbidimetric and kinetic chromogenic assays.
The gel clot assay is a semi-quantitative approach to endotoxin detection. It requires an hour-long incubation period at 37°C and provides a visual means of confirming the presence of pyrogenic factors in the form of gelation or clotting. The simplicity and ease of use of this method make it an attractive option. This test is also less prone to false results as fewer factors can interfere with the process. However, the process also has a fairly long preparation time, is not quantitative and is not particularly amenable to automation4.
Endpoint chromogenic tests measure endotoxin levels photometrically. Samples are combined with LAL and incubated at 37°C with a chromogenic substrate. In the presence of endotoxins, a yellow colour that absorbs light at 405nm develops. This can be measured and the endotoxin concentration calculated using a standard curve.
Kinetic turbidimetric assays are often used to test water or large volume parenterals, e.g. saline fluids for drip administration. Samples are incubated at 37°C with LAL, and the change in turbidity is assessed over time by measuring the absorbance at 340nm. The higher the level of endotoxins in the sample, the faster it becomes cloudy. Hence, the reaction time of the assay is inversely proportional to endotoxin concentration, allowing a sample to be quantitatively analysed with the aid of a standard curve.
One of the most sensitive tests currently available is the kinetic chromogenic LAL assay, which is commonly used when testing antibiotics and vaccines. It is a combination of the colourimetric and kinetic assays described above and has a broad sensitivity range.
Regardless of which assay method is used, each can be inhibited or enhanced by additional factors present in a sample, much of which can be overcome by pre-treating samples prior to testing. In most cases pharmaceutical regulatory authorities do permit pre-treatment, but only if the process in use is validated using a spike-in control5. One simple but effective method to overcome interference is to dilute samples in LAL reagent water. However, in some cases additional treatments are required:
pH LAL assays should be carried out within the pH range specified in the test protocol. Typically this is between pH 6.0 and 8.0. Care should be taken to adjust the pH where necessary, as pH values outside this range can impact the effectiveness of LAL assays.
Chelators Samples containing strong chelating agents will sequester the divalent cations usually used by the active enzymes underpinning the LAL assay. Diluting samples in a divalent cation solution, such as MgCl2, will likely overcome this problem.
Aggregates Endotoxins are composed of hydrophilic polysaccharides and hydrophobic lipids, the latter of which are targeted by the LAL reagent. In some cases, endotoxin monomers aggregate to ‘shield’ their hydrophobic lipid portions from the aqueous LAL solution, ‘hiding’ them from the enzymes in the LAL assay. To overcome this challenge, metallo-modified polyanionic dispersing detergents and surfactants can be used to trigger aggregate dissociation.
All of the LAL assays described are reliant on the biological product of the horseshoe crab. Although it has proven a powerful approach for detecting endotoxins, it does suffer from weaknesses. Due to the biological nature of LAL, utilising blood from the horseshoe crab introduces lot-to-lot variability. Differences between the blood of animals, seasonal changes and environmental factors can all contribute to the variability of the final product. It is also prone to non-specific beta (1, 3)-glucan activity, which can mimic the clotting and colour alterations that are used to indicate the presence of endotoxins4.
Fortunately, recombinant DNA technology has enabled the development of alternative assays that are not reliant on the horseshoe crab. These make use of a recombinant endotoxin-sensitive protein, which works in combination with a fluorogenic substrate to activate a specific protease cascade and trigger a quantitative readout. The process can be used to detect endotoxin levels across a wide range of concentrations with high sensitivity (0.005-5 endotoxin units/ml). A global, multi-centre study recently demonstrated that the recovery of endotoxins from water and other tested products using a recombinant endotoxin-sensitive protein assay was comparable to that of LAL-based methods. The results of the assay validation were published in the Pharmacopeial Forum in 20106.
Endotoxin testing is an invaluable tool across a range of research, pharmaceutical and bioproduction applications.
The effective use of LAL tests and newer, recombinant assays facilitates the effective detection of endotoxins, reducing the risk of illness and death when manufacturing medicinal products. Reliable endotoxin tests also enable researchers to carry out efficient, effective and insightful cell culture experiments, with a greatly reduced fear of contamination.
References:
1. Bang, F.B., 1956. A bacterial disease of Limulus Polyphemus. Bull. Johns Hopkins Hosp., 98, pp. 325-351.
2. Levin, J. and Bang, F.B., 1964. A description of cellular coagulation in the Limulus. Bull. Johns Hopkins Hosp., 115, pp. 337.
3. Levin, J. and Bang, F.B., 1964. The role of endotoxin in the extracellular coagulation of Limulus blood. Bull. Johns Hopkins Hosp., 115, pp. 265–274.
4. Prior, R.B., 1990. The Limulus Amoebocyte Lysate Test. In: R.B. Prior ed., Clinical Applications of the Limulus Amoebocyte Lysate Test. Florida: CRC Press. pp. 27-36.
5. Council of Europe, 2010. 2.6.14 Bacterial Endotoxins. In: European Pharmacopoeia 7.0. Strasbourg: European Department for the Quality of Medicines within the Council of Europe. pp. 171-175.
6. Pharmacopeial Forum Vol. 36(1) [Jan.–Feb. 2010], Stimuli to the Revision Process: A Recombinant Factor C Procedure for the Detection of Gram-negative Bacterial Endotoxin.
The author:
Holly Kabrick is Product Manager at Lonza Walkersville
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