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That is consistent with mine, for whatever one more account is worth.
Analytics · Impurities & related substances · continued
Endotoxin and why it is a separate question from purity — what changed since posts 31–60
This is a continuation of a long topic, addressed by post number rather than by page. Start at post 1.
k.agyeman, post #11: 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. Go to post
Post #29 is right about the mechanism and I think understates the practical bit.
The honest reading of a purity figure: it is an upper bound on how much of what the method could see was the intended species, under one integration convention, on one sample.
This has been discussed before and I could not find the thread, so, again.
Reframing Endotoxin slightly, because I think the disagreement is about the question rather than the answer. If the question is "does it happen", yes. If it is "how often", nobody here knows.
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Deletion sequences (incomplete coupling during synthesis): lower in mass by one residue. Chromatographically they usually elute earlier or later depending on the residue's hydrophobicity. They are the most common impurity in solid-phase synthesis.
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Confirming post #33 from a second method, which matters more than confirming it from a second person.
Residual solvents: traces of solvents used in purification. These are usually tested by gas chromatography, not by HPLC. A specification for residual solvents should be stated separately from the purity.
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j.sorensen, post #1: 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… Go to post
Related substances: compounds chemically related to the target peptide but not the target peptide itself. The standard method separates them and reports them as area percent. How related they can be before they exceed specification is a regulatory question.
Posting it because the silence on this was starting to look like agreement.
Worth stating the null on Endotoxin before we explain it: the observation may be nothing. That possibility deserves a sentence and usually does not get one.
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Nardone, post #42: Dimer and higher-order multimers: two or more peptide molecules bonded together. They appear at double the mass and higher. They may or may not separate from the monomer on HPLC depending on the method. Go to post
What I would want before treating Endotoxin as settled: the method, the sample, and whether anyone tried to find the opposite result. Two of the three are usually missing.
m.onwuka, post #9: 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. Go to post
Noted, and I have changed what I was going to do on the strength of it.
Endotoxin is well covered in the tag pages, and the older discussions are better than the recent ones because they were argued out properly. Worth twenty minutes before adding to this one.
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Deamidation at asparagine or glutamine adds approximately one dalton and frequently produces a close-eluting pair. It is the impurity most likely to be integrated into the main peak by accident.
The literature is thinner on this than the confidence in the thread implies.
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Building on post #46 rather than restating it.
Taking Endotoxin seriously for a moment rather than deflecting: the honest position is that the community has observations and no controlled comparison, and those two things support very different sentences.
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j.hartmann, post #15: 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. Go to post
Post #45 put the caveat in the right place and I want to underline it.
Marking my uncertainty on Endotoxin explicitly. I am confident about the direction, much less confident about the size, and not confident at all that it generalises past the case in the first post.
Confirming post #46 from a second method, which matters more than confirming it from a second person.
Aggregates: multiples of the monomer mass. May not elute at all under a standard reversed-phase method. A species that does not come off the column does not appear in the area percentage.
That is all I can say without guessing.
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k.ogunleye, post #43: What I would want before treating Endotoxin as settled: the method, the sample, and whether anyone tried to find the opposite result. Two of the three are usually missing. Go to post
I had written a reply contradicting post #48 and deleted it. Here is what survived.
Where I have landed on Endotoxin, having got it wrong once in public: the direction is clear, the magnitude is not, and anyone quoting a precise magnitude has borrowed it from somewhere that did not measure it.
l.vermeulen, post #47: Building on post #46 rather than restating it. Taking Endotoxin seriously for a moment rather than deflecting: the honest position is that the community has observations and no controlled comparison, and those two things support very different sentences. Go to post
The arithmetic in post #51 is right; the assumption feeding it is the part to check.
An update on my earlier Endotoxin post: the pattern held for another six weeks and then stopped, which I did not predict and cannot explain.
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Post #51 put the caveat in the right place and I want to underline it.
Endotoxin sits at the boundary between what this community can usefully discuss and what it cannot, and I think it falls on the discussable side, narrowly.
Residual solvents: traces of solvents used in purification. These are usually tested by gas chromatography, not by HPLC. A specification for residual solvents should be stated separately from the purity.
That is all the detail I have. Someone else will have more.
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Adding a null result on Endotoxin. I looked, carefully, and found nothing, and null results deserve posting precisely because they never are.
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p.lindqvist, post #45: Endotoxin is well covered in the tag pages, and the older discussions are better than the recent ones because they were argued out properly. Worth twenty minutes before adding to this one. Go to post
A supplier that can tell you what its principal impurity is has answered a harder question than one that can tell you its purity, and the answer is more useful.
Post #55 is the version of this I will quote in future. One addition.
The strongest argument against my own position on Endotoxin, stated as well as I can state it, since nobody else has yet.
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I read post #55 twice before replying, because I had assumed the opposite.
Peptide impurities that differ by a single residue are the hardest to resolve and the most likely to be biologically relevant, which is an unfortunate combination.
I keep a log of this specifically because memory is unreliable about it.
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Endotoxin looks different depending on whether you are reading the primary literature or the summaries of it, and the difference is not in our favour.
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