Storage and stability of lyophilised peptides

Most peptide degradation that reaches us as a complaint did not happen in transit or in our freezer. It happened in the customer's laboratory, through a small number of avoidable mechanisms. This is what they are and what to do about them.

Why the lyophilised form is so much more stable

Almost every degradation route available to a peptide needs water: hydrolysis of the backbone, deamidation at asparagine and glutamine, and the conformational mobility that lets oxidation-prone side chains find oxygen. Freeze-drying removes the water and the reactions slow by orders of magnitude. This is why lyophilised material carries a shelf life in years and a solution carries one in weeks.

Storage conditions that actually matter

• Temperature. −20 °C for lyophilised material is sufficient for the stated shelf life. −80 °C is better for long-term storage and for anything already in solution.
• Moisture. Keep the vial sealed and let it equilibrate before opening. If you are storing an opened vial, a desiccant in the box is worth the small effort.
• Light. Relevant for tryptophan- and tyrosine-containing sequences in particular. We supply light-sensitive compounds in amber vials; keep them in the box regardless.
• Oxygen. Methionine and cysteine oxidise readily. For sequences containing either, an inert headspace on an opened vial is worthwhile.

The vial looks empty

This is the most common support question we receive and it is almost always a non-issue. Two to five milligrams of lyophilised peptide forms a film that can be genuinely difficult to see, and material is routinely thrown onto the vial walls and stopper during transit. Centrifuge briefly, or tap the vial down firmly on the bench, before adding solvent. The peptide content figure on the certificate is what confirms mass — visual inspection confirms nothing.

Once it is in solution

This is where most avoidable loss occurs, and freeze–thaw cycling is the leading cause. Each cycle concentrates solutes at the ice boundary, drives local pH shifts as buffer components crystallise at different rates, and promotes aggregation. Peptide lost to aggregation does not come back on the next thaw.

• Aliquot on first reconstitution, into volumes matched to a single experiment. This one habit prevents more degradation than every other measure combined.
• Use low-binding tubes for dilute solutions. At low microgram-per-millilitre concentrations, adsorption to polypropylene can remove a substantial fraction of what you weighed out.
• Add solvent slowly down the vial wall, and swirl rather than vortex. Shear and foaming both promote aggregation at the air–liquid interface.
• Write the reconstitution date on the tube. When a result eventually looks wrong, it is the first thing you will want and the thing nobody ever recorded.

Working out what you actually have

A vial labelled 5 mg does not contain 5 mg of peptide. It contains 5 mg of lyophilised solid, of which some is acetate counter-ion and some is residual water. With typical figures — 8% acetate, 4% water — the peptide content is nearer 88%, and the certificate states it directly from amino acid analysis.

For most qualitative work the difference is immaterial. For anything where concentration has to be right — a dose–response curve, an IC50, a quantitative binding measurement — use the peptide content figure from the certificate rather than the label mass. It is the difference between a result you can publish and one you cannot reproduce.