Post #2 describes the usual case. This is about the unusual one. 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.
Posting this under the heading it deserves: Revisiting: Aggregates and why a purity assay may miss them entirely Everything below is what sits behind that.
I would like to disagree carefully with the settled view on Aggregates, and I would like to be argued out of it if the disagreement is bad.
The disagreement is about one step, not about the conclusion. If the step holds I withdraw it entirely.
Adding the measurement that the opening post says would settle it.
Aggregates is a question about a distribution, not about a value, and treating it as a value is what produces the confident wrong answers.
Post #2 describes the usual case. This is about the unusual one.
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.
Adding a reference point for Aggregates. Mine is a single case, collected without controls, and I am posting the method alongside it so it can be discounted appropriately.
Grateful for the specificity. Vague answers to this question are what sent me looking.
The arithmetic in post #4 is right; the assumption feeding it is the part to check.
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.
Genuine question rather than a rhetorical one: has anyone here actually observed Aggregates, as opposed to read about it? The thread is long and I cannot tell.
Post #8 answers the question as asked. The question underneath it is different.
Truncation products: fragments from incomplete synthesis or from degradation. They elute quite differently from the intact peptide because they are much smaller and have different hydrophobicity. They are usually well separated.
The honest answer on Aggregates is that it depends, and the useful part is the list of what it depends on. Four items, in rough order of how much they matter.
Most people get the first two right and then argue about the fourth.
Two people in this thread mean different things by Aggregates and are disagreeing about the definition while believing they are disagreeing about the facts. Worth pausing to define it.
Nothing to add, except that this is the answer I would give if asked.
Post #12 is the version of this I will quote in future. One addition.
The version of Aggregates that circulates here is a simplification of a simplification. It is not wrong, but it has lost the conditions under which it holds, and those conditions are where the interesting cases live.
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.
Taking Aggregates 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 #14 put the caveat in the right place and I want to underline it.
Agreed on Aggregates, with one qualification that I think matters. The reasoning holds for the case as described. Change the starting assumption and it does not, and the starting assumption is the part nobody states.
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.
I have said this before in a thread nobody could find, so it is worth repeating.
I would call the community position on Aggregates likely rather than established, and I would be comfortable defending that hedge.
Picking up post #17: that is the part I would want checked first.
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.
I have deliberately not rounded that, because the rounding is where the argument starts.
Summarising the Aggregates thread so far, since it is long and the answer is buried: the first reply has the method, the fourth has the correction to it, and the rest is people agreeing at length.
Taking post #22 at face value and following it one step further.
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.
If anyone has run this properly I would rather read that than my own guess.
That matches what I have seen, for whatever a single anecdote is worth.
Post #22 is right about the mechanism and I think understates the practical bit.
Aggregates may be a multiple of the monomer mass and may not elute at all under a standard method. What does not come off the column does not appear in the area percentage.
None of the above is medical advice and I am not qualified to give any.
Coming back to post #25, because the follow-up matters more than the original answer.
The failure mode on Aggregates is boring rather than dramatic. It is almost always the step everyone assumes was done correctly because it is too simple to get wrong.
Since Aggregates 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.
I have no financial interest in anything named in this thread and I want to say so before I comment on Aggregates, because it is the sort of subject where it matters.
That last point is the ceiling on what any purity figure can claim. A method that cannot see a species cannot exclude it, and no certificate says which species its method cannot see.