Endotoxin testing for research peptides: LAL methods and recombinant factor C

Endotoxin is lipopolysaccharide (LPS) released from Gram-negative bacteria. It is heat-stable, survives ordinary autoclaving, and is not reliably removed by the filters that remove bacteria themselves, so a sterile-filtered material can still carry endotoxin. Glassware is depyrogenated with dry heat instead, commonly 30 minutes at 250 °C. In laboratory work, endotoxin contamination can confound cell-culture and other biological assays, which is why some researchers ask for it.

LAL is an extract of amebocytes (blood cells) from horseshoe crabs (Limulus polyphemus, or Tachypleus tridentatus for some reagents). Endotoxin triggers an enzyme cascade in the lysate that ends in a clot, which the gel-clot method reads directly. Turbidimetric and chromogenic methods measure the same cascade with an instrument and compare the sample against a standard curve made from a reference endotoxin.

Recombinant factor C replaces the lysate with the first enzyme of that cascade made by recombinant DNA technology, so no horseshoe-crab blood is used. Ph. Eur. chapter 2.6.32 describes a test with recombinant factor C. Because it responds to endotoxin through a single enzyme, it is not affected by the beta-glucan pathway that can give false positives with some LAL reagents.

Peptides and buffers can inhibit or enhance the reaction, so a raw number is not enough. The pharmacopeial test requires a check that the sample, at the dilution used, does not interfere.

1. Method and reagent: gel-clot, kinetic turbidimetric, chromogenic, or rFC, with the reagent sensitivity (for gel-clot, the labeled lambda in EU/mL).
2. Units tied to the material: EU per mg of peptide or EU per mL of a stated solution, not a bare 'pass'.
3. Dilution: the dilution tested, which must stay within the maximum valid dilution for the stated limit.
4. Interference control: a positive product control (spike recovery). For quantitative methods USP <85> expects recovery between 50% and 200%.
5. Lot and date: the same lot number as the vial and the date of testing.

Our currently published lot reports cover HPLC purity, measured quantity, and on some lots LC-MS identity. They do not include endotoxin results. We say that plainly so nobody assumes a screen that was not run. If your work needs endotoxin data for a specific lot, ask through the testing page before ordering and we will tell you whether a report exists.