Endotoxin Testing and Removal
Background
Endotoxin normally refers to the lipopolysaccharides existing in the outer cell membrane of gram-negative bacteria. High amounts of endotoxin can cause fever, inflammations, septic shock, and even death after entering the blood circulation. However, in the manufacturing of biopharmaceutics, endotoxin contamination is common. For example, in the process of using E. coli to express recombinant protein nanocages, endotoxin of E. coli is released in cell disruption and can contaminate protein nanocages. In addition, because of the wide existence of bacteria in water and the environment, and the possibility of random endotoxin release in cell growth and division, endotoxin contamination may occur in almost every step in preparation. Endotoxin is resistant to heat and extreme pH. Conventional sterilization cannot fully deactivate it. The activity level of endotoxin in injectables such as vaccines and drugs is strictly limited by Food and Drug Administration for the sake of safety. Hence, endotoxin activity detection and endotoxin removal are crucial in the development of protein nanocages as vaccines or drug delivery platfroms.
Fig 1. Gram-negative Bacterial Cell Membrane Structure and Endotoxin Location (Alexander, C., Rietschel, E., 2001)
Protein nanocages are an emerging candidate in vaccine development and drug delivery. Prior to animal tests, endotoxin activity in purified protein nanocages must be under 5 EU/kg. Creative BioMart Nanocage provides highly-sensitive, FDA-approved service for endotoxin qualitative and quantative analysis. We cover various endotoxin removal techniques so as to find an effective procedure to remove endotoxins without affecting protein nanocage stability and conformation.
Endotoxin Testing
Our endotoxin testing service includes the bacterial endotoxin/limulus amebocyte lysate (LAL) test and rabbit pyrogen test. LAL test is conducted in vitro. LAL is a horseshoe crab blood cell extract sensitive to endotoxin. At the presence of endotoxin, the enzymes in LAL are activated and a cascade of reactions are triggered, leading to the clotting of LAL. Rabbit pyrogen test is an animal test. Briefly, it is conducted by intravenously injecting the sterile sample into healthy rabbits and measuring the increase of rabbit body temperature.
Table 1. Endotoxin tests
| Endotoxin test | Usages and features |
|---|---|
| LAL test | Well-suited for qualitative and quantitative analysis of endotoxin activity. Gel clot, kinetic chromogenic, and kinetic turbidimetric methods are available. More specific, time-efficient, and sample-saving than rabbit pyrogen test. |
| Rabbit pyrogen test | Applicable in a wider range of pyrogen detection, such as endotoxin and pyrogens from gram-positive bacteria. |
Endotoxin Removal
Endotoxin removal is usually integrated into the protein purification pathway. If endotoxin activity is still high after removing protein impurities and nucleic acids, an additional endotoxin removal step is necessitated. Current endotoxin removal methods are grounded in different mechanisms and the procedure of endotoxin removal needs to be tailored to suit the specific target protein, due to the diversity of protein samples. For instance, anion exchange chromatography is suitable for cationic proteins, but not for anionic proteins. Ultrafiltration is efficient in separating endotoxin aggregates but not in the cases of protein nanocages. Affinity absorbent and aqueous two-phase micellar system are another two widely-used methods.
Creative BioMart Nanocage has rich experiences in all purification techniques and owns well-equipped laboratories to conduct all endotoxin removal approaches. Our endotoxin removal approaches include:
- Endotoxin affinity chromatography
- Anion exchange chromatography
- Ultrafiltration and size-exclusion chromatography
- Aqueous two-phase micellar system (Triton X-114-based)
We guarantee a low endotoxin activity (< 0.01 EU/µg) and a high protein recovery after endotoxin removal. Our service consists of only 3 steps, 1) the analysis of target protein to select the best-suited endotoxin removal approach, 2) endotoxin removal experiment conduction, and 3) delivery of the endotoxin-free sample and the result report.
If you are interested in our endotoxin testing and removal service, please do not hesitate to contact us through online inquiry.
References
- Alexander, C., Rietschel, E. (2001). "Bacterial lipopolysaccharides and innate immunity." Endotoxin Res, 7,167-120.
- Silva, F., et al. (2021). "Combined Method to Remove Endotoxins from Protein Nanocages for Drug Delivery Applications: The Case of Human Ferritin." Pharmaceutics, 13(2), 229.
