Freeze-Thaw Cycles and Aliquoting: Protecting Peptide Stock Solutions

What happens when a solution freezes

When a solution freezes, water leaves as ice and everything dissolved in it is pushed into the shrinking volume of liquid between the ice crystals. The solutes become far more concentrated than they were, which is why studies of freezing refer to the effect of concentrated solutes on the sample.^{[1, 2]} The way freezing is carried out also affects the structure and quality attributes of the frozen and dried material.^[3] A peptide in that concentrated liquid is exposed to higher concentrations of itself, of salts and of buffer than it ever saw before freezing.

The buffer itself can change. In sodium phosphate, the less soluble disodium salt crystallizes out during freezing, and the pH of the frozen solution fell from 7.0 to as low as 3.8 in one model protein study, while the same experiment in potassium phosphate shifted the pH by at most 0.3 units.^[1] Inactivation of the protein tracked the pH change. Whether a buffer protects or harms a frozen sample therefore depends on which salt it is made from. See buffer and pH in the glossary.

The rate of freezing and thawing matters

Using model enzymes in dilute solution, researchers found that slow freezing combined with fast thawing gave the best recovery of activity, and fast freezing combined with slow thawing the worst. Fast freezing makes small ice crystals with a large ice–liquid interface that proteins can be damaged against, and during slow thawing recrystallization adds further stress.^[2] In the same study, when buffer salts took part in freezing, recovery of activity fell because the pH of the buffer changed during freezing.^[2]

These were enzymes, not short peptides, and the precise rates will not transfer. The transferable point is that a freeze-thaw cycle is a physical event with its own variables, so two stocks frozen differently are not equivalent.

What published data show about repeated cycles

Two studies on different molecules illustrate both the risk and its dependence on conditions.

In recombinant MCP-1, a protein, solutions kept at 4 °C for seven days lost no significant amount. At −20 °C and −81 °C the first freeze-thaw cycle contributed some loss that depended on concentration and time, and the second cycle caused approximately 50% loss. The authors concluded that multiple freeze-thaw cycles should be avoided.^[4]

In substance P, a peptide, six freeze-thaw cycles in tubes pre-coated with bovine serum albumin left more than 75% of the radioactivity recoverable after the last cycle, whereas recovery was minimal in uncoated or siliconized glass tubes.^[5] The same study found that the peptide stored in Tris buffer at pH 7.4 underwent peptide-bond cleavage, which was not seen in a triethylamine formate buffer at pH 3.14.^[5]

Taken together, they show that loss on freezing is not a fixed percentage. It depends on the molecule, the buffer and the container, and a number from one system is not a number for another.

Aliquoting: one thaw per portion

An aliquot is a measured portion of a larger solution. Dividing a stock solution into aliquots before it is first frozen means that each portion is thawed once, and a portion that is not used is never put through a second cycle. That design follows directly from the data above: if the second cycle costs material, the way to avoid it is not to have one.

Aliquot size is a planning question and a piece of arithmetic. Decide how much solution one use needs, divide the stock into that many equal portions, and keep a small surplus. The concentration calculator gives the amount of material in an aliquot from a concentration and a volume you enter, and reconstitution math sets out the equations.

Container surfaces count too

Peptides adsorb to the walls of tubes and vials, and the amount depends on the peptide and the surface. In a study of eight radiolabelled peptides covering a wide range of charge, size and end groups, recovery after 48 hours differed between glass and plastic tubes, whether untreated or siliconized; siliconizing decreased recovery, whereas adding bovine serum albumin improved it.^[6] The authors concluded that the container should be chosen by testing for the peptide in question, because peptide loss to surfaces can be unpredictable.^[6]

Albumin is not always acceptable in an assay, so the practical lesson is narrower: do not assume that a tube is neutral, and compare containers if a result depends on the concentration being right. See adsorption in the glossary.

A checklist for a frozen stock

1. Choose the number of aliquots before freezing. Base it on the number of uses planned, plus a small margin.
2. Choose the container deliberately. Use the same type for every aliquot and test recovery if the concentration is critical.^[6]
3. Label every aliquot. Concentration, diluent, date prepared and aliquot number.
4. Thaw each aliquot once. Treat a thawed aliquot that is not used as used. The loss seen on a second cycle in published data is the reason.^[4]
5. Check against a fresh preparation. Where it matters, compare a thawed aliquot with a freshly made solution by HPLC, looking at peak area and any new peaks.
6. Record the history. The storage and temperature log has a freeze-thaw record for this purpose.

Reviews of protein stability treat physical instability, such as aggregation, alongside chemical instability and discuss how the two are interrelated; the chemical routes are covered in storage stability and degradation pathways.^[7]