How many freeze-thaw cycles will cagrilintide at 6.67 mg/mL tolerate?

Details up front: cagrilintide · 6.67 mg/mL.

I would like to understand the steps well enough to explain them to someone else.

I have access to a refrigerator with a logger and a freezer without one, which may be relevant.

So: what is the actual procedure, and which steps matter as opposed to being ritual?

Nobody has published a cycle count, and at 6.67 mg/mL the concentration tells you what a wrong guess costs: a 0.1 mL aliquot holds 0.67 mg and every microlitre is 6.67 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 6.67 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 6.67 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.

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.

Degradation pathway by condition

Thaw slowly at room temperature or in the refrigerator rather than in warm water. Rapid warming creates local thermal and concentration gradients that promote aggregation.

The caveat is that aggregation is invisible in a clear solution below the threshold where it becomes visible.

Aliquot before the first freeze. That is the whole answer.

Start with how many cycles are actually planned, because one or two are immaterial and ten are not.

Let a frozen vial reach room temperature before opening it. Opening a cold vial in humid air condenses water into the cake, which raises residual moisture and undoes what lyophilisation achieved.

A dry lyophilised powder is much less affected by a temperature cycle because there is no liquid phase for anything to concentrate into. Condensation on a cold vial opened warm is the real risk there.

Cryoconcentration of solutes at the ice front is a well-documented mechanism in freeze-thaw damage to proteins and peptides.

Aliquoting itself is a handling step and introduces its own contamination opportunity.

Dry powder tolerates cycles far better than solution does.