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Endotoxins and their Relevance in R&D ApplicationsLPS Detection & Removal Methods

Contaminations of cell cultures show severe problems in the research and production industry. The attention turns here particularly to the prevention of any contamination with bacteria, Mycoplasmas, yeast or fungi. For companies that develop substances for therapeutic applications endotoxins are nowadays a main focus.

Endotoxins (Lipopolysaccharides, LPS) are biologically active, structural components of the outer cell membrane of all Gram-negative bacteria. Contaminations of biological solutions (e.g. plasmid DNA, peptide, rec. proteins, antibody) with LPS lead to artefacts and misinterpretations during high-sensitive stimulation experiments (cell cultures or animal models). Therefore prevention, removal & detection of LPS get more and more important1.

Characteristics of LPS
Endotoxine have a hydrophilic polysaccharide and a lipophilic lipid part. Unlike bacteria LPS are very heat and pH stable. Moreover, the amphipatic LPS molecules tend to form large aggregates (> 106 Dalton) in aqueous solutions. Due to the high hydrophobicity they are also very affine to other hydrophobic substances.

LPS & Immune system
The biological activity of endotoxin is associated with the LPS. Toxicity is associated with the lipid component (Lipid A) and immunogenicity is associated with the polysaccharide components. Therefore LPS mediates a variety of proinflammatory and toxic functions which are responsible for much of the pathology in servere infections like sepsis and septic shock. LPS causes a very strong stimulation of different cells of the immune system, such as monocytes, macrophages, B cells, polymorphic nuclear- and endothelial vascular cells.

LPS is biologically effective even in lowest concentrations (lower pg/ml range). They will be released during cell lyses but unlike enterotoxines or exotoxines they will not be continuously released in the culture media from living bacteria.

TLR4 is the receptor for LPS from Gram-negative bacteria. However, TLR4 alone is not sufficient to confer LPS responsiveness. TLR4 requires MD-2, a secreted molecule, to functionally interact with LPS. Furthermore, a third protein, called CD14, was shown to participate in LPS signalling, leading to NF-kB translocation. This signalling is mediated through the adaptor protein MyD88 but also through a MyD88-independent pathway that involves the (TIR) domain-containing adapter protein (TIRAP)4 5 6.

LPS & Cell culture
Endotoxins have, depending on the cell type and culture conditions, an influence on cell growth, cell differentiation, contractility and protein expression pattern. They trigger the release of tumor necrosis factor (TNF), Interleukins-1,6 and 8 and the production of Platelet-activating factors, Prostaglandin E2, Thromboxane A27 8 .

LPS Detection
Today the LAL (Limulus Amebocyte Lysate) assay is used for the routine check of LPS in biological solutions. It was introduced on the market in the seventies and replaces more and more the rabbit pyrogenic test which was used since 1940. These tests were the first detection methods that had been certificated by the FDA.

In the meantime the humane full blood test (EAA, Endotoxin Activity Assay9) has been also given admittance by the FDA for the identification of patients with high sepsis risk.

Both, the FDA & US Pharmacopoeia regulate that medical equipment or drugs, which enter the circulating blood or the central nervous system, may not cause any pyrogenic reactions. Therefore everything declared as "pyrogenic free" has to be checked.

The advantage of the rabbit test (Ph. Eur. 2002, 2.6.8) is that all known and unknown pyrogenics can be detected. Therefore it is indispensable for the drug study till now. But it represents a timeconsuming animal experiment and not all substances that seem pyrogenic do cause fever in rabbits. In the LAL test (Ph. Eur. 2002, 2.6.14) a special property of the haemolymph of Limulus polyphernus is used: in the presence of LPS it starts to coagulate.

The following LAL test methods were developed meanwhile:
Gel Clot
Chromogenic End-Point Test
Kinetic Turbidimetric Test
Kinetic Chromogenic Test

The LAL test has the advantage that it is sensitive and can be better standardized than the rabbit test. Moreover, it allows a quantitative test evaluation. However it is very susceptible and it is also an animal experiment thats why it can be transferred only with restriction to the human system. A further disadvantage is that only unbound LPS molecules can be detected. Therefore it can be possible that up to 30-50 lower values10 are measured with the LAL test than with detection systems which consider only the chemical connection and not the molecule activity. Thus there are different new approaches for the LPS detection. In the indirect method (e.g. "monocyte activation cytokine assay") the endotoxins are detected via mediators (e.g. II 1 , II 6, TNF-a). Thereby mainly monocytes or leukocytes like RAW264.7, MonoMac 6 or THP 111 are used.

The in-vitro pyrogenic test (IPT) is a patented procedure to detect pyrogenic determination and is based on humane whole blood. It is an alternative for the rabbit test since it detects also positive bacteria and others biological pyrogenics (such as exotoxines, lipoteichoic acid, parasitical constituents) which cannot be verified in the LAL.

They are also methods which use the 3-Hydroxy fatty acids of the lipopolysaccharides for the endotoxine detection via gas chromatography/mass spectrometry.

The LPS concentration is indicated in EU/ml or EU/mg. The unit EU (endotoxin unit) describes the biological activity of LPS. 1 EU is generally converted to 100 pg LPS.

A Gram-negative bacteria contains approximately 10-15 g of LPS, 1 EU therefore can be generated by only 105 bacteria.

LPS Removal
Removal of endotoxin is one of the most difficult downstream processes during protein purification. Many commercial available products are unable to remove endotoxin satisfactorily, or require time consuming incubation steps. In many cases, complete endotoxin removal is only achieved with massive substrate loss. Reduction or removal of endotoxin to less than 1 ng/mg (10 EU/mg) is a very difficult task. Only methods with highest endotoxin removal capacity combined with excellent recovery rates of the target substance are reasonable and acceptable. To meet exactly these most challenging requirements, Profos AG has developed an endotoxin trap for lab- and pilot-scale: EndoTrap.

* Th e regeneration substance is NOT (sodium) deoxycholate! DOC would have cytotoxical effects on cell culture and also influence the cell growth and the morphology of the cells.

LPS removal with EndoTrap
EndoTrap is an affinity matrix for the efficient removal of bacterial endotoxins from solutions. The protocol of EndoTrap is user friendly, yields rapid results as it is designed as a flow-through system and does not require training or special equipment. EndoTrap is applicable also for the downstream process as it is available as 50% slurry, too. The EndoTrap resin can also be performed on fully automated liquid chromatography systems. Therefore EndoTrap connects the research and the production field.

If you are interested in detailed information about the EndoTrap-family please visit our website at In the download section you can find the EndoTrap publications, product descriptions and our FAQs.

Profos offers an endotoxin removal and also an endotoxin detection service (using a quantitative, kinetic chromogenic LAL assay). Please inquire for our service. If you liked to learn more about our products and "gene 2 protein" services, please visit our website (section Services).

Customer Feedback
EndoTrap, our endotoxin removal and endotoxin detec tion services serve the needs of scientist of the bioresearch and bioprocess markets thereby helping to avoid artefacts and misinterpretation caused by endotoxin contamination when performing highly sensitive stimulation experiments in cell culture or animal models.

Here are some statements from our customer regarding EndoTrap or Profos AG:
... "probably the best system of recent years"
"It is a very good product"
"easy to use, can be used in any lab without additional equipment"
"very good, clearly a product that was needed in the market"
"good helper for removing endotoxin"
"fast and efficient method"
"very effective"
"good product, would recommend it to other colleagues"
"it works, its convenient, the service is excellent"
... "It is also worth mentioning the adequate, competent, and immediate support from Profos AG team. The way of interaction is very straight forward, uncomplicated, and solution oriented."

Dr. Stephanie Steck
Head of Products for R&D / Endotoxin Group (M&S)
Profos AG
Josef-Engert-Str. 11
93053 Regensburg

fon: +49-941-942 6226
fax: +49-941-942 6220


Profos AG

1. Hanrock, R.E.W. Bacterial outer membranes: evolving concepts. Specific structures provide gram-negative bacteria with several unique advantages. AS M News 1991; 57: 175-82.

2. Profos Endotoxin Compendium

3. Profos Endotoxin Compendium

4. Poltorak, A. et al. (1998) Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282(5396):2085-8.

5. Shimazu, R. et al. (1999) MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4. J. Exp. Med. 189(11):1777-82.

6. Horng, T., Barton G.M. and Medzhitov, R. (2001) TIRAP: an adapter molecule in the Toll signalling pathway. Nat. Immunol. 2(9):835-41.

7. Rietschel, E.T. and Brade, H. (1992) Bacterial Endotoxins. Sci. Amer. 267:54-61.

8. Ulmer, A.J., Flad, H., Rietschel, T. and Mattern, T. (2000) Induction of proliferation and cytokine production in human T lymphocytes by lipopolysaccharide (LPS). Toxicology 152 (1-3):37-45

9. Spectral Diagnostics Inc. Endotoxin Activity Assay.

10. Rylander R. Endotoxin in the environment - exposure and effects. J Endotoxin Res. 2002;8(4):241-52.

11. Hartung, et al. ECVAM workshop on novel pyrogen tests based on the human fever reaction. ATLA 29, 99-123, 2001.



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