Aggregates and why a purity assay may miss them entirely Writing it up because I had to work it out twice and would rather nobody else did.
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.
On aggregates, 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.
The opening post answers the question as asked. The question underneath it is different.
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.
Sensible. I would want the same detail before I acted on it either.
Narrowing post #5, because the general version has more than one answer.
Relative response factors mean impurities are not detected in proportion to how much of them is present. A one per cent peak is not one per cent by mass unless the response factors happen to match.
I would treat that as a working assumption and revisit it.
Comparing impurity profiles across suppliers is much more informative than comparing purity figures, and almost nobody has the documents to do it.
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.
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.
Worth separating two things that post #12 runs together.
I have been on both sides of the aggregates argument in this category within eighteen months, which should tell you how strong the evidence for either side is.
Appreciated. The plain phrasing does more work here than a longer post would.
Coming back to post #12, because the follow-up matters more than the original answer.
The number people quote for aggregates is a central estimate presented without its interval, and the interval is wide enough that the estimate is nearly uninformative on its own.
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.
Caveat: everything above assumes the paperwork is what it says it is.
Answering the question post #16 raises rather than the one it answers.
A definition problem is doing most of the work in this aggregates discussion. Once the term is pinned down I suspect the disagreement mostly goes away and what is left is small.
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.
I would be interested in a counterexample if anyone has one.
Adding the measurement that post #20 says would settle it.
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.
Anyone who has looked at this more carefully, please correct the record.
Where I part company with post #22, and it is a narrow parting.
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.
What I would check first on aggregates is whether the thing being measured moved or whether the way of measuring it moved. Those look identical in a graph.
This is the first time the answer has come with its own limits attached. Appreciated.
Scavengers and cleavage-cocktail residues can persist and appear as small early-eluting peaks. They are process-related rather than sequence-related, which is a useful distinction when reading a profile.
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 holds for the case as described. Change the assumptions and it may not.
Comparing impurity profiles across suppliers is much more informative than comparing purity figures, and almost nobody has the documents to do it.
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.