Deamidation and the close-eluting pair it produces Writing it up because I had to work it out twice and would rather nobody else did.
Something about deamidation does not reconcile and I would like a second pair of eyes before I decide which half is wrong.
Two sources, both reputable, giving figures that cannot both be right unless they are measuring different quantities. My suspicion is that they are, and I cannot see how.
Narrowing the opening post, because the general version has more than one answer.
A request rather than an answer: could whoever has the primary source for deamidation post it? I have seen the claim three times this month and each version had lost a qualifier.
Noted, and thank you for writing it out rather than summarising it.
I disagree with the framing of deamidation above, and I think it is a substantive disagreement rather than a terminological one. Setting out why, so it can be checked.
The reasoning depends on an assumption that is doing a lot of work and is never stated. If the assumption holds, the conclusion follows. I do not think it holds generally.
I read the opening post twice before replying, because I had assumed the opposite.
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.
The general answer and the answer for your case may diverge here.
Post #5 answers the question as asked. The question underneath it is different.
Comparing impurity profiles across suppliers is much more informative than comparing purity figures, and almost nobody has the documents to do it.
Adding this to the thread rather than to the wiki, because I am not confident enough for the wiki.
Two things can be true about deamidation at once: the mechanism is plausible and the evidence for the size of the effect is thin. Most of the argument here is people defending the first against attacks on the second.
I keep a log for deamidation specifically because my memory of it turned out to be systematically wrong in one direction. Six weeks of notes cost nothing and settled it.
Oxidation at methionine or tryptophan adds sixteen per oxygen and typically elutes earlier. In aged material it is the modification that grows.
Adding the measurement that post #9 says would settle it.
Before the thread moves on from deamidation — 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.
Taking post #10 at face value and following it one step further.
Taking deamidation 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.
Post #9 and I disagree about the size of the effect, not about the direction.
Oxidation at methionine and tryptophan: adds 16 per oxygen. Usually elutes earlier. Oxidation is common in storage, especially if the solution is exposed to light or if antioxidants are not present.
The answer changed when I changed how I was measuring, which was informative.
Sensible. I would want the same detail before I acted on it either.
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.
Building on post #14 rather than restating it.
A methods point on deamidation rather than a substantive one: if the comparison is not like for like, the difference you are measuring is the difference in method.
Two people in this thread mean different things by deamidation and are disagreeing about the definition while believing they are disagreeing about the facts. Worth pausing to define it.
Deamidation at asparagine and glutamine: adds 1 approximately. Frequently appears as a close-eluting pair. It is a chemical modification that occurs during storage.
Everything in post #16 holds. The case it does not cover is the one I have.
I have no financial interest in anything named in this thread and I want to say so before I comment on deamidation, because it is the sort of subject where it matters.
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.
Take the reasoning and check the arithmetic; I do not always get it right.
Genuine question rather than a rhetorical one: has anyone here actually observed deamidation, as opposed to read about it? The thread is long and I cannot tell.
Building on post #21 rather than restating it.
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.
That is a description of practice, not a recommendation of it.
Everything in post #21 holds. The case it does not cover is the one I have.
The most useful reply I ever got about deamidation was a request to state my units. It sounds like pedantry and it has saved me twice.
Since deamidation keeps coming up, it should probably be a maintained page rather than a recurring thread. I am happy to draft it if someone with more direct experience will review it.
Adding a note of thanks rather than an opinion. I did not know most of that.
I read post #25 twice before replying, because I had assumed the opposite.
Checked the deamidation claim against the primary source this morning. It survives, with a narrower scope than the version quoted here. Posting the narrower scope.
Post #25 answers the question as asked. The question underneath it is different.
Reporting rather than recommending, on deamidation. What happened is above. Whether it should have is a different question and not one I am qualified to answer.