Accepted answer
Nobody has published a cycle count, and at 1 mg/mL the concentration tells you what a wrong guess costs: a 0.1 mL aliquot holds 0.1 mg and every microlitre is 1 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 1 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 1 mg/mL and the date, and never thaw a container you will refreeze.
The short version: aliquot before freezing, thaw slowly, never refreeze a thawed aliquot, and count your cycles.
The mitigation is aliquoting. Divide the reconstituted solution into single-use volumes before the first freeze, and each aliquot then experiences exactly one cycle.
Degradation pathway by condition
| Pathway | Dominant when | Detected by |
|---|
| Deamidation | Solution, neutral to alkaline pH | RP-HPLC, +1 Da on MS |
| Oxidation | Light, trace metals, peroxides | RP-HPLC, +16 Da on MS |
| Hydrolysis | Solution, extremes of pH | RP-HPLC, fragment masses |
| Aggregation | Agitation, interfaces, high concentration | SEC, visual haze; often invisible on RP-HPLC |
| Freeze-concentration damage | Freeze-thaw of buffered solution | SEC, loss of recovered content |
Never refreeze a thawed aliquot. The whole point of aliquoting is that the aliquot is single-use, and refreezing it discards the benefit.
Cryoconcentration of solutes at the ice front is a well-documented mechanism in freeze-thaw damage to proteins and peptides.
Dry powder tolerates cycles far better than solution does.
edited 17 Aug 2024 by h_pergande — fixed an arithmetic slip in the third paragraph
The doubling-per-ten-degrees rule is the part I did not know and now use constantly. – sian_llewellyn 5 days ago 2Does the same reasoning apply to material already in solution, or is that a different curve? – pk_curve 2 months ago add a comment