On the subject in the title: Endotoxin and why it is a separate question from purity — what changed since Working notes rather than a conclusion.
What changes if the standard account of Endotoxin is wrong? I ask because I have been treating it as settled and I noticed this week that I could not say why.
Working through the consequences rather than the evidence, since others here are better placed on the evidence.
Worth separating two things that the opening post runs together.
Endotoxin has a well-known answer and a correct answer, and the interesting work is establishing that they are the same. Nobody has done that here yet.
The claim about Endotoxin upthread is stronger than its source supports. I have read the source. The source says "associated with" and the post says "causes".
The bit of Endotoxin that nobody enjoys is that the answer changes depending on what you are trying to decide with it. Say what the decision is and the thread will converge.
Freeze-drying itself does not generate impurities in any significant way. Handling before and after it does, which is why transit conditions belong in the discussion.
I would want a second opinion before relying on that.
Deletion sequences arise from incomplete coupling and are lower by one residue mass. Where they elute depends on the hydrophobicity of the residue that is missing, so they can appear on either side of the main peak.
If you are new and reading this thread for the answer to Endotoxin: the answer is conditional, the conditions are in the third reply, and the rest of the thread is worth skipping.
Source for the Endotoxin figure, since it was asked for. It is in the discussion rather than the abstract, which is why the version circulating is stronger than the paper is.
Reading the surrounding paragraph is worth the two minutes. The authors are more careful than their summarisers.
Disulfide formation: if a peptide contains cysteine, it can form disulfide bonds with itself or with other molecules. Under oxidising conditions multiple species appear. Reducing conditions (like DTT) convert them back.
Post #11 and I disagree about the size of the effect, not about the direction.
Racemisation produces a diastereomer that is mass-identical and chromatographically resolvable only on a method chosen for the purpose. Standard reversed-phase frequently will not separate it.
Taking post #11 at face value and following it one step further.
The useful distinction on Endotoxin is between what was measured and what was inferred from it. Both end up in the same sentence and only one of them has error bars.
What would change my mind on Endotoxin is a second dataset collected by someone with no stake in the first. Until then I hold it loosely and I would rather say so than pretend to more.
Post #15 answers the question as asked. The question underneath it is different.
Off-target structures: if the sequence synthesis goes wrong, a completely different amino acid can be incorporated. The resulting off-target peptide is a structural isomer with the same mass but a different sequence. No chromatographic purity method detects this without a reference standard.
Worth separating two things that post #15 runs together.
Two claims get bundled together under Endotoxin and they need separating. The descriptive one — this is what was observed — is usually well supported. The causal one — this is why — usually is not.
Almost every disagreement in threads like this one dissolves once you say which of the two you are making.
Reading rather than contributing, but this is the most useful thread I have found on it.
Post #15 describes the usual case. This is about the unusual one.
Comparing impurity profiles across suppliers is much more informative than comparing purity figures, and almost nobody has the documents to do it.
Before the thread moves on from Endotoxin — what is the sample size behind the claim? I am not being difficult; I have seen the same figure quoted from an n of four and from an n of four hundred.
Acknowledging rather than arguing. The reasoning holds as far as I can follow it.
The related-substances total is only as informative as the method that generated it. Two per cent by a method that resolves everything is a different statement from two per cent by a method that resolves little.
For what it is worth, the same held on the two occasions I checked.
I had written a reply contradicting post #22 and deleted it. Here is what survived.
On Endotoxin the community has more anecdote than the confidence in this thread implies, and I include my own contribution in that.
Endotoxin: I have looked for the primary source twice and failed twice. Either it does not exist or it is somewhere I do not know to look, and I would like to know which.
Incomplete deprotection: mass higher by the protecting group mass. Usually markedly later eluting. A synthesis artifact from incomplete removal of protecting groups.
A guess, clearly labelled as one.
On Endotoxin, I would rather understate and be corrected upward than overstate and be quoted. That is a house style here and it is a good one.
Whatever the answer on Endotoxin turns out to be, the method for getting there is the same: state the assumption, do the arithmetic in public, invite the correction.