Lyophilization, or freeze-drying, removes water from a frozen material by sublimation: under vacuum, the ice turns directly to vapor without passing through a liquid phase. It runs in three stages, freezing, primary drying and secondary drying, and leaves a porous dry solid called a cake. Research peptides are supplied lyophilized because a dry peptide is far more stable than the same peptide in solution.
This post explains the physics behind the process, what happens in each stage, why water removal protects a peptide, and how to read the cake in a vial before you open it.
The physics: why ice can skip the liquid phase
Water has a triple point, the one combination of temperature and pressure at which ice, liquid water and vapor coexist in equilibrium. It sits at 0.01 °C (273.16 K) and a vapor pressure of 611.657 Pa, a value measured at the US National Bureau of Standards in 1976. Below that pressure, liquid water cannot exist at equilibrium: warm ice and it goes straight to vapor.
Lyophilization uses that fact. The material is frozen solid, the chamber is pumped down to a small fraction of the triple-point pressure, and gentle heat is supplied to the ice. The vapor travels to a condenser held far colder than the product, where it freezes out again. What remains is a dry solid that keeps the shape and volume of the frozen fill, full of fine channels where ice crystals used to be.
FDA's inspection guide on lyophilization describes the process in the same terms: water "is removed from a product after it is frozen and placed under a vacuum, allowing the ice to change directly from solid to vapor without passing through a liquid phase", in three separate, interdependent stages.
The three stages at a glance
| Stage | What happens | Typical conditions | Water removed |
|---|---|---|---|
| Freezing | Water crystallizes as ice; solutes concentrate between crystals | Product cooled to about −40 °C | None; water is immobilized as ice |
| Primary drying | Ice sublimes under vacuum | 50 to 200 mTorr (about 7 to 27 Pa); product held below its collapse temperature | The frozen (ice) fraction |
| Secondary drying | Bound water desorbs from the solid | Shelf warmed, often to 30 to 50 °C | The unfrozen, bound fraction |
The conditions are drawn from Tchessalov and colleagues' 2023 review of freeze-drying process design. Every product gets its own settings. These are orders of magnitude, not a recipe.
Stage 1: freezing
The solution is filled into vials, placed on temperature-controlled shelves, and cooled. Water does not freeze at exactly 0 °C: it supercools until ice nucleates, then crystallizes rapidly. As pure ice forms, the peptide and any other solutes are squeezed into the shrinking spaces between the crystals, a state called freeze concentration.
What that concentrated phase does next determines how the rest of the process must run.
- Crystalline solutes (mannitol and glycine, for example) can crystallize out, forming a rigid scaffold. A crystalline system can be dried at a relatively warm product temperature.
- Amorphous solutes (sugars such as sucrose and trehalose, and most peptides) do not crystallize. They form a glass, and the key property is its glass transition temperature in the freeze-concentrated state, written Tg′. Above Tg′ the glass softens.
Some processes include an annealing step, holding the frozen product somewhat warmer, typically −15 to −10 °C for a few hours, so that small ice crystals grow into larger ones. Larger crystals leave larger channels, which lets vapor escape faster in the next stage. The review recommends a final product temperature of about −40 °C before drying begins.
Stage 2: primary drying
Primary drying is sublimation, and it is usually the longest part of the process.
The chamber is evacuated, typically to 50 to 200 mTorr, and the shelves are warmed just enough to supply the heat that sublimation consumes. A drying front moves down through the frozen fill: above it is dry, porous solid, below it is still ice. Water vapor leaves through the dry layer and is trapped on the condenser.
The critical constraint is temperature. If the product at the drying front gets warmer than its collapse temperature (Tc), the softened solid can no longer hold up its own structure and the pores fuse. For amorphous material, Tc usually lies a few degrees above Tg′. The review advises holding the product several degrees below Tc, with a wider safety margin for shorter runs.
Running too cold wastes time, because sublimation rate rises steeply with temperature. Running too warm collapses the cake. Process design is largely the search for the warmest safe product temperature.
Stage 3: secondary drying
When the ice is gone, the solid still holds water that never froze: water bound to the peptide and trapped in the glassy matrix. It leaves by desorption, not sublimation, which needs warmth rather than low pressure alone.
The shelves are ramped up, often to 30 to 50 °C, slowly for amorphous products so the matrix does not soften faster than it dries. Tchessalov and colleagues report that secondary drying can be completed in 3 to 6 hours at a shelf temperature of 40 or 50 °C, with water content below 0.5% by weight commonly achieved. A 2023 review of drying technologies cites a residual moisture of 3% or less as the ceiling for maintaining protein stability.
At the end, the vials are usually stoppered inside the chamber, under vacuum or an inert gas, before air is let back in. FDA's inspection guide notes that stoppering is typically done by hydraulic or screw-driven mechanisms built into the lyophilizer.
Why dry peptides are more stable
Most of the chemistry that degrades a peptide needs water, either as a reactant or as the medium that lets molecules move and meet.
| Degradation route | Where it happens in a peptide | Role of water |
|---|---|---|
| Hydrolysis | The backbone, especially at aspartate-glycine bonds | Water is a reactant |
| Deamidation | Asparagine and glutamine side chains, fastest at Asn-Gly and Asn-Ser | Fastest in neutral or basic solution |
| Oxidation | Methionine and cysteine side chains | Dissolved oxygen and mobility speed it up |
| Aggregation | Hydrophobic stretches of sequence | Needs molecular mobility |
A 2023 review of peptide and protein instability puts it plainly: thermostability is often markedly improved when the material is dry. The same review notes an important exception. The succinimide intermediate on the path to deamidation does not require water, so deamidation can still proceed slowly in the solid state. Dry is slower, not frozen in time. That is why lyophilized peptides are still stored frozen, and why the guide to storing peptides treats temperature, light and moisture together.
Lyophilization also changes nothing about the molecule's sequence or identity. It removes solvent. The purity measured by HPLC on the dry powder, covered in how HPLC measures peptide purity, describes the same material that went into the vials.
Reading the cake before you open the vial
A well-dried cake is a single, uniform, porous plug with the volume of the original fill. It may be white or off-white, and may have pulled slightly away from the glass. Several defects are visible without opening anything:
Collapse. The structure has shrunk or fused, often from the bottom up, because the product ran above its collapse temperature during drying. Collapsed material can hold more residual water.
Meltback. FDA's guide defines meltback as a form of collapse caused by a change from solid to liquid in the vial, that is, incomplete sublimation. It looks like a glassy or dissolved layer at the base.
Shrinkage and cracking. Minor pulling away from the wall is common and usually cosmetic. Heavy shrinkage alongside a glassy surface is not.
Discoloration or a sticky film. A change from the lot's normal color, or a wet-looking residue, suggests moisture uptake or degradation.
Anhydrolabs leaves about 3/8 in of bare glass on every label so the cake can be checked without removing anything. The storage and handling page sets out what to do with a cake that has collapsed, melted back or changed color: send the order number and the product, and it will be replaced.
Handling lyophilized material in the lab
Dry is the state a vial ships in. It is not a property the powder keeps once the seal is broken, because a lyophilized cake is porous and many peptides are hygroscopic.
- Store it frozen and dark. Anhydrolabs recommends −4 °F (−20 °C), away from light, from the day it arrives.
- Warm before opening. Let the vial reach room temperature before breaking the seal, so moisture in the air does not condense on cold powder.
- Reconstitute only what you need. Once dissolved, the stability advantage of the dry state is gone. The reconstitution guide covers preparing in-vitro stock solutions, and reconstituted material holds for about four weeks at 39 °F (4 °C).
- Aliquot instead of refreezing. Repeated freezing and thawing of one solution exposes it to ice-interface stress each time.
How Anhydrolabs supplies lyophilized peptides
Every Anhydrolabs compound, for example TB-500, is supplied as a lyophilized powder in a vacuum-sealed vial, and also as 10-vial kits. Each vial carries its lot number, and the certificate of analysis for a lot is available from [email protected]. The material is for in-vitro laboratory research only, as set out in the research-use statement and explained further in what research use only means.