Accepted answer
Nobody has published a cycle count, and at 5 mg/mL the concentration tells you what a wrong guess costs: a 0.1 mL aliquot holds 0.5 mg and every microlitre is 5 µg. Damage from freezing is not gradual attrition — it is concentrated at the phase transitions, where ice excludes solute and the unfrozen fraction climbs well above 5 mg/mL for as long as the transition lasts. Two slow cycles can therefore do more than four fast ones, which is why a cycle count is the wrong unit in the first place. The way to make the number one is to make it one: split at reconstitution into single-draw aliquots, label each with 5 mg/mL and the date, and never thaw a container you will refreeze.
This is one of the few handling questions with a genuinely quantitative literature behind it.
Never refreeze a thawed aliquot. The whole point of aliquoting is that the aliquot is single-use, and refreezing it discards the benefit.
During freezing, solutes are excluded from the ice lattice and concentrate into the residual liquid. Local concentrations can rise many-fold, which promotes aggregation independently of temperature.
Selective crystallisation of sodium phosphate buffer components producing large pH shifts on freezing is a classical result in the lyophilisation literature.
Dry powder tolerates cycles far better than solution does.
edited 13 Mar 2026 by Dr_Otto_Lindqvist — updated for the 2026 guidance change
4Adding a vote because this deserves more of them. – n_takahashi 6 months ago 3Does the same reasoning apply to material already in solution, or is that a different curve? – anouk_desmet 4 months ago add a comment