Endotoxin and Sterility Testing: What the Numbers Mean
Sterility and endotoxin are routinely treated as one item on a checklist. They are unrelated tests answering unrelated questions, and a lot can pass one convincingly while failing the other. If your material is going anywhere near cell culture, the distinction is not academic.
Sterility asks whether anything is alive
USP <71> sterility testing incubates the sample in growth media across two conditions — one favouring aerobic bacteria and fungi, one favouring anaerobes — for fourteen days, and looks for turbidity. The result is binary: growth or no growth.
It is a test of viable organisms at the moment of testing. It says nothing about whether organisms were present earlier in the process and were subsequently killed by filtration, lyophilisation or the low water activity of a dried cake.
Endotoxin asks what dead bacteria left behind
Endotoxin is lipopolysaccharide from the outer membrane of Gram-negative bacteria. It is a molecule, not an organism, and it is extraordinarily heat-stable — standard autoclaving does not destroy it, and it passes through the 0.22 µm filters that produce a sterile solution.
This is the crux: a preparation can be perfectly sterile and heavily contaminated with endotoxin, because sterilisation removed the bacteria and left their membrane fragments behind. That is not a hypothetical failure mode, it is the normal one.
Reading EU/mg
The Limulus amebocyte lysate assay quantifies endotoxin in endotoxin units. Reported per milligram of peptide, EU/mg lets you calculate the endotoxin load your assay will see at your working concentration.
Cell culture is where this bites. Endotoxin activates TLR4 and drives inflammatory signalling at concentrations well below anything that affects viability, so a contaminated lot does not kill your cells — it quietly adds an inflammatory stimulus to every condition. If you are measuring cytokine output, NF-κB activity or anything downstream of innate immune signalling, endotoxin contamination does not add noise, it adds a confound that points in a consistent direction.
Our release specification is under 5.0 EU/mg and typical measured values run below 1.0. The measured figure, not just the pass, is on every certificate.
Where contamination comes from
Water is the usual source. Endotoxin accumulates in water systems and standard purification will not remove it, which is why depyrogenated water and glassware are separate specifications from sterile ones. Raw materials, resins and column packings contribute; so does handling.
The practical implication for your own bench work is that your diluent matters. Reconstituting a low-endotoxin lot with ordinary laboratory water can reintroduce more endotoxin than the peptide ever carried.
Put it into practice
Every Certus lot has a public certificate you can audit against everything above, before you spend anything.