At 4 °C the question is which route is fastest, not whether aggregation happens — and the routes do not share an activation energy, so their ranking changes with temperature. 4 °C is the condition the rule of thumb is anchored to, so it is the baseline rather than a multiplier: everything else in this thread is quoted relative to it. That multiplier is an average over every route at once, which is exactly why it cannot tell you which one wins. Molecules associate without any covalent change, so the mass is unchanged and a reversed-phase run — which is performed in organic solvent — mostly dissolves the evidence before it can be measured. So the way to answer it for your vial is to pick the method that sees aggregation specifically and run it against a control held cold, rather than to infer a mechanism from a purity number that averages all of them.
The honest answer is that most reported "degradation" is adsorption and dilution error rather than chemistry.
Aggregation is physical: peptides unfold at air-liquid interfaces and associate. Shaking maximises that interface, which is why swirling and shaking produce visibly different outcomes on the same vial.
It helps to be literal here: deamidation converts asparagine or glutamine to the corresponding acid via a succinimide intermediate, adding one dalton. It is base-catalysed, accelerates above neutral pH and is the dominant aqueous pathway for many peptides.
Nothing here is medical advice, and research-use compounds are not approved for human use.
Swirl, never shake. Aggregation is a handling problem more than a time problem.
3Same experience here, different supplier. – Dr_Elias_Weiss 14 days ago 2I would add a sentence about light, since tryptophan-containing sequences care. – yuki_morishita 9 months ago add a comment