In plain English
Endotoxin is a piece of the outer wall of certain bacteria (Gram-negative bacteria, such as E. coli). It is also called lipopolysaccharide, or LPS. It is released from bacterial cells, so it can be present even when no living bacteria are left [1, 5].
Very small amounts of endotoxin strongly switch on immune cells. They do this through a receptor called TLR4 (a sensor on the cell surface) [2]. That is a problem in laboratory experiments. If a sample contains endotoxin, cells may react to the endotoxin rather than to the molecule being studied [3].
The main test uses an extract from horseshoe crab blood, called LAL. Endotoxin makes it clot or change colour. A newer test uses a lab-made version of the same crab protein, called recombinant factor C [1].
Results are given in endotoxin units (EU). A low EU value on a certificate shows that endotoxin was measured and found to be low in that batch.
The short version
- Endotoxin comes from bacteria but is not alive.
- Sterility testing and endotoxin testing are different tests.
- LAL and rFC are the main methods.
- EU measures activity, not mass.
What endotoxin is
Endotoxin is lipopolysaccharide, a major component of the outer membrane of Gram-negative bacteria [1, 2]. It has three parts: lipid A (the part recognised by the TLR4 receptor complex), a core sugar region, and a variable O-antigen chain [2]. In solution, endotoxin forms aggregates and micelles, and it binds to many surfaces and molecules, including proteins [5].
Endotoxin is a frequent contaminant of solutions produced in bioprocesses, and filtration methods used to decontaminate water have little effect on endotoxin levels in protein solutions [5]. It can enter a synthetic peptide through water, reagents, equipment or handling.
Why endotoxin matters in the laboratory
Minute amounts of LPS can start potent innate immune responses. The LPS-binding protein, CD14 and the TLR4–MD-2 receptor complex recognise the lipid A pattern [2]. Cells such as macrophages and monocytes are especially responsive.
This creates a well-documented source of false findings. In one study, commercial recombinant heat shock protein 60 had been reported to make macrophages release the inflammatory signal TNF-α. A highly purified preparation with low endotoxin activity did not do this at concentrations up to 10 µg/mL, while a less purified preparation did. Removing LPS and LPS-associated molecules essentially eliminated the activity [3].
For any experiment involving immune cells, primary cells or cytokine readouts, an unmeasured endotoxin level is a confounding variable. Endotoxin has also been found in products sold online: in a 2024 study of semaglutide products bought without prescription, every tested sample contained endotoxin [8].
How the LAL test works
The Limulus amebocyte lysate (LAL) test uses an extract of blood cells (amebocytes) from the horseshoe crab. The lysate contains a cascade of serine proteases. Endotoxin activates factor C, the first enzyme in the cascade, which ends in a gel clot [1]. The LAL test is the most pharmacopoeia-recognised technique for endotoxin detection [6].
US guidance describes three LAL method types, set out in USP chapter <85> (Bacterial Endotoxins Test) [7]:
| Method | What is measured | Output |
|---|---|---|
| Gel clot | Whether a firm clot forms at a defined lysate sensitivity | Pass/fail at a limit, or a semi-quantitative value by dilution series |
| Turbidimetric (kinetic) | Increase in cloudiness as the clot forms | Quantitative EU value from a standard curve |
| Chromogenic (kinetic or end-point) | Colour released from a synthetic substrate cleaved by the cascade | Quantitative EU value from a standard curve |
A valid test also includes controls. A known amount of endotoxin is added to a portion of the sample (a “positive product control”) to show that the sample itself is not inhibiting or enhancing the reaction [7].
Recombinant factor C (rFC)
Recombinant factor C is a laboratory-produced version of the horseshoe crab enzyme that starts the LAL cascade. It was developed partly because of variation in LAL sensitivity and specificity and concern about the supply of horseshoe crabs [1]. rFC assays do not require crab blood [1].
In a comparison using LPS from several bacterial species, an rFC assay detected most LPS structures in picogram quantities. Potency was similar to LAL assays for some LPS types and different for others. Only the rFC assay correlated significantly with IL-6 release from a monocytic cell line in that study [4].
Endotoxin units (EU)
An endotoxin unit is a unit of biological activity, not of mass. It is assigned by comparison with a reference standard endotoxin. Different endotoxin preparations have different activity per nanogram. In one collaborative study, an E. coli O55:B5 control standard endotoxin lot contained approximately 4.5 EU per ng [9]. There is no single fixed conversion between EU and nanograms.
How to read EU on a Certificate of Analysis:
| Reported as | Meaning |
|---|---|
| EU/mg | Endotoxin activity per milligram of material. The most useful form for a lyophilised peptide |
| EU/mL | Endotoxin activity per millilitre of solution; depends on concentration |
| ”< 0.5 EU/mg” | Below the limit of quantitation reported by that method on that batch |
| “Pass” without a number | Met an unstated specification; less informative |
Worked example: from EU/mg to the assay well
A batch COA reports 0.5 EU/mg. A stock solution is made at 1.0 mg/mL, then diluted 1 in 100 into cell-culture medium (final peptide concentration 10 µg/mL).
- Endotoxin in the stock: 0.5 EU/mg × 1.0 mg/mL = 0.5 EU/mL
- Endotoxin in the well: 0.5 EU/mL ÷ 100 = 0.005 EU/mL
Whether that level matters depends on the cell type and readout. Running a parallel vehicle control and, where possible, an endotoxin-only control makes the contribution visible.
Why tests can under-read: low endotoxin recovery
Some sample matrices hide endotoxin from the test. In low endotoxin recovery (LER), endotoxin added to a product cannot be fully measured after a hold time. A review of LER groups the reported causes into three areas: the endotoxin standards used in hold-time studies, the protein active ingredient, and the excipients [6]. A method should therefore be checked for the specific sample type.
Endotoxin is not sterility
| Test | Detects | Does not detect |
|---|---|---|
| Endotoxin (LAL/rFC) | Endotoxin from Gram-negative bacteria, alive or dead | Living organisms; Gram-positive bacteria or fungi |
| Sterility | Living microorganisms that can grow in culture media | Endotoxin from dead bacteria |
| Bioburden | Number of viable microbes before sterilisation | Endotoxin |
Sterile filtration (0.22 µm) is designed to remove bacteria; it is not an endotoxin-removal step. Even ultrafiltration, used to decontaminate water, has little effect on endotoxin levels in protein solutions [5]. Sterile technique during reconstitution reduces new contamination but does not remove endotoxin already present.
What this means in Australia
- Not on the ARTG. Pharmacopoeial endotoxin testing is a requirement set for regulated medicines and devices [7]. Unapproved peptide products are not entered on the Australian Register of Therapeutic Goods (ARTG) and have not been assessed by the TGA [10].
- An endotoxin result does not change status. A low EU value is evidence about one batch’s quality. It does not change a substance’s Poisons Standard status; several peptides are named in Schedule 4 (prescription-only) and Appendix D, clause 5 (possession without authority is illegal) of the current Poisons Standard (October 2026) [11].
- Labels don’t change status. The TGA has stated that a “research use only” disclaimer does not make supply lawful, change regulatory status, permit importation, or remove advertising or supply obligations [10].
- Accredited testing. NATA accredits Australian testing laboratories to ISO/IEC 17025, which is a useful signal of method control for an endotoxin report [12].
This is general information, not legal advice. Laws change; check the current Poisons Standard and TGA guidance.
Frequently asked questions
What is an endotoxin test?
It measures bacterial endotoxin (lipopolysaccharide) in a sample, usually with the LAL test or recombinant factor C. Results are reported in endotoxin units (EU) [1, 7].
What does EU/mg mean on a COA?
It is the endotoxin activity per milligram of material. Because EU is a unit of activity, it cannot be converted to nanograms with a single fixed factor [9].
Does sterile filtering remove endotoxin?
No. A 0.22 µm filter is designed to remove bacteria, not endotoxin. No single method removes endotoxin from every protein solution, and methods used for water have little effect on protein solutions [5].
Why does endotoxin matter for in vitro research?
Trace endotoxin activates immune cells through TLR4, so it can produce effects that are wrongly attributed to the molecule under study [2, 3].
What is the difference between LAL and rFC?
LAL is an extract of horseshoe crab blood cells containing a full enzyme cascade. rFC is a recombinant version of the first enzyme in that cascade and does not require crab blood [1, 4].
References
- Ding JL, Ho B. Endotoxin detection—from limulus amebocyte lysate to recombinant factor C. Subcell Biochem. 2010;53:187–208. doi:10.1007/978-90-481-9078-2_9. PMID: 20593268
- Park BS, Lee JO. Recognition of lipopolysaccharide pattern by TLR4 complexes. Exp Mol Med. 2013;45(12):e66. doi:10.1038/emm.2013.97. PMID: 24310172
- Gao B, Tsan MF. Recombinant human heat shock protein 60 does not induce the release of tumor necrosis factor alpha from murine macrophages. J Biol Chem. 2003;278(25):22523–22529. doi:10.1074/jbc.M303161200. PMID: 12686536
- Abate W, Sattar AA, Liu J, Conway ME, Jackson SK. Evaluation of recombinant factor C assay for the detection of divergent lipopolysaccharide structural species and comparison with Limulus amebocyte lysate-based assays and a human monocyte activity assay. J Med Microbiol. 2017;66(7):888–897. doi:10.1099/jmm.0.000510. PMID: 28693666
- Petsch D, Anspach FB. Endotoxin removal from protein solutions. J Biotechnol. 2000;76(2–3):97–119. doi:10.1016/s0168-1656(99)00185-6. PMID: 10656326
- Cao Y, Zhang Y, Qiu F. Low endotoxin recovery and its impact on endotoxin detection. Biopolymers. 2021;112(11):e23470. doi:10.1002/bip.23470. PMID: 34407207
- US Food and Drug Administration. Guidance for industry: Pyrogen and endotoxins testing: questions and answers. fda.gov. Accessed 11 October 2026.
- Ashraf AR, Mackey TK, Vida RG, et al. Multifactor quality and safety analysis of semaglutide products sold by online sellers without a prescription: market surveillance, content analysis, and product purchase evaluation study. J Med Internet Res. 2024;26:e65440. doi:10.2196/65440. PMID: 39509151
- Pearson FC, Weary ME, Sargent HE, et al. Comparison of several control standard endotoxins to the National Reference Standard Endotoxin—an HIMA collaborative study. Appl Environ Microbiol. 1985;50(1):91–93. doi:10.1128/aem.50.1.91-93.1985. PMID: 3896144
- Therapeutic Goods Administration. Understanding your responsibilities when importing, compounding and supplying unapproved peptide products. Safety advisory, 13 April 2026. tga.gov.au. Accessed 11 October 2026.
- Therapeutic Goods (Poisons Standard—October 2026) Instrument 2026 (F2026L01327). Federal Register of Legislation. legislation.gov.au. Accessed 11 October 2026.
- National Association of Testing Authorities, Australia (NATA). Accreditation programs, including ISO/IEC 17025 testing and calibration. nata.com.au. Accessed 11 October 2026.
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