What is Lyophilisation?
Also called: Lyophilization, Freeze-drying
Freeze-drying: removing water from a frozen solution by sublimation under vacuum, leaving a dry, porous solid that keeps far longer than the same peptide in solution.
By the APL Research Team · Updated
Lyophilisation removes water from a peptide solution without letting it melt: the solution is frozen, then the ice is turned straight into vapour under vacuum. What remains is the white cake or powder in a peptide vial. Because most degradation reactions need water as a reactant or a medium, the dry solid is far more stable than a solution, which is why purified peptides are finished and shipped this way.
The three stages
- Freezing. The solution is cooled until water crystallises as ice, concentrating the peptide and any excipients into the spaces between crystals. How ice nucleates and crystallises at this step affects the rest of the process and the quality of the final product [1].
- Primary drying. Chamber pressure is dropped below water's triple point (611 Pa at 0.01 °C), so ice sublimes rather than melting. The product must stay below its collapse temperature, which is related to the glass transition of the freeze-concentrate (Tg′); above it the structure softens and slumps [1].
- Secondary drying. The shelf is warmed to desorb water still bound to the solid, bringing residual moisture down further [1].
Dry is not inert
A review of solid-state stability lists deamidation, peptide bond cleavage, oxidation, the Maillard reaction, β-elimination and dimerisation or aggregation as reactions that still occur in dried peptides and proteins, governed mainly by temperature, moisture content, excipients and whether the solid is amorphous or crystalline [2]. Moisture is the factor most under the bench's control. Lyophilised vials are therefore kept cold (−20 °C for long holds) and sealed, and a vial taken from the freezer is left to reach room temperature before opening so that condensation does not land on the cake.
Reading the cake
| Appearance | Usual meaning |
|---|---|
| Intact white puck | Normal cake |
| Loose powder or fragments on the walls | Cake broken up in transit; common with small fills |
| Thin film or very little visible material | Normal for 5–10 mg of peptide with no bulking agent |
| Shrunken, glassy or sticky residue | Collapse during drying or moisture uptake; worth querying with the supplier |
Worked example: water is part of the fill
Residual and absorbed moisture count towards the weighed mass. If a 10 mg cake held 5% water by weight, 0.5 mg of it would be water, before counter-ions are considered. That is one reason net peptide content sits below the gross fill, and one reason it can fall over time if a vial repeatedly takes up humidity from being opened cold.
What happens after the vial is opened
The stability advantage ends at reconstitution. Solutions are held at 2–8 °C for short periods and divided into single-use aliquots for frozen storage. Freezing a solution in an ordinary freezer is not lyophilisation: the water stays in the tube, and every thaw is another freeze–thaw cycle, with the ice–liquid interfaces and concentration effects that can drive aggregation.
The lyophilisation guide covers process design in more depth, and the peptide storage guide the practical conditions for dry and dissolved material.
References
- 1.Tang X, Pikal MJ. Design of freeze-drying processes for pharmaceuticals: practical advice. Pharm Res. 2004. PubMed 15032301
- 2.Lai MC, Topp EM. Solid-state chemical stability of proteins and peptides. J Pharm Sci. 1999. PubMed 10229638