Storage science for lyophilized material
Freeze-drying slows a peptide’s chemistry; it does not stop it. Three things set the pace at which dry material changes: temperature, light and moisture. This page covers dry, sealed material only, and explains the science behind the usual rule: keep it cold, dark and dry.
Schematic
Text version of this figure
| Driver | What it does | Answer |
|---|---|---|
| Heat | Speeds up every chemical reaction. | Cold |
| Light | Drives oxidation of residues that absorb it. | Dark |
| Moisture | Supplies water for hydrolysis and deamidation, and loosens the solid so molecules can move and meet. | Dry |
What “change” means
Change in a stored peptide is specific: particular chemical reactions turn some of it into slightly different molecules, which then appear as impurities. The main ones are:
- Oxidation. Sulfur-containing and aromatic residues react with oxygen. Methionine becomes methionine sulfoxide, 16 Da heavier; cysteine can form disulfide bonds; tryptophan can also be oxidized.
- Deamidation. An asparagine side chain loses ammonia by way of a ring-shaped intermediate, a succinimide, and ends up as aspartate or its isomer, isoaspartate. The mass rises by about 1 Da. It is fastest where asparagine is followed by glycine.
- Hydrolysis. Water breaks a peptide bond, cutting the chain in two.
- Aggregation. Molecules stick to one another.
Many of these show up as new peaks in HPLC or as mass shifts in mass spectrometry. Peptide manufacturers’ handling guides note that sequences containing asparagine, glutamine, methionine, cysteine or tryptophan tend to have shorter shelf lives, which fits this chemistry.
Temperature
Chemical reactions run faster when warm. The relationship is described by the Arrhenius equation, and a common rule of thumb drawn from it is that a reaction rate rises by a factor of about two to three for every 10 °C increase. Run the arithmetic the other way and cooling is powerful. If a reaction doubles with every 10 °C, then moving it from 25 °C to 5 °C slows it about fourfold, and moving it to −20 °C slows it roughly twenty-fold. With a factor of three, the same moves slow it about ninefold and more than a hundredfold.
Schematic
Text version of this figure
| Storage temperature | Relative rate (doubling rule) | Relative rate (tripling rule) |
|---|---|---|
| 25 °C | 1 | 1 |
| 5 °C | 1/4 | 1/9 |
| −20 °C | about 1/23 | about 1/140 |
That is arithmetic from a rule of thumb, not a prediction for any particular material. Real reactions vary, and dry solids can depart from simple behavior, especially near their glass transition temperature, the point at which a glassy solid starts to soften and its molecules gain freedom to move. Moisture lowers that temperature, which is one reason temperature and humidity work together.
This is why peptide manufacturers’ handling guides commonly call for freezer storage, around −20 °C or colder, for dry peptide kept over the long term, with colder generally preferred.
Light
Ultraviolet light, and to a lesser extent visible light, can drive chemical reactions directly. A residue that absorbs light enters a reactive state and can then be oxidized; this is photo-oxidation. The residues that undergo it directly include tryptophan, tyrosine, phenylalanine and cysteine. Opaque or amber packaging and dark storage remove the trigger.
Moisture
Freeze-dried peptide is hygroscopic: it pulls water from the air, and its porous structure gives it a large surface to do so (see Why research peptides ship as a powder). Absorbed water causes three problems:
- it is a reactant, for hydrolysis and deamidation;
- it is a plasticizer, loosening the solid so molecules can move and meet;
- it adds mass, so a given mass of powder holds less peptide than before.
One easy way for moisture to get in is condensation. When a cold container is opened in warm room air, the air touching the cold surfaces cools below its dew point and water condenses, on the container and on the powder inside. That is why peptide manufacturers’ handling guides say to let a cold, sealed container reach room temperature before it is opened for analysis or sampling, ideally inside a desiccator, a closed box kept dry by a drying agent. Tight closures, and a desiccant in the outer packaging, keep humid air away while the material is stored.
Oxygen
Oxidation needs oxygen. Sequences prone to it, especially those with cysteine, methionine or tryptophan, are best kept with as little contact with air as possible: a well-sealed container with little empty space above the material. Some handling guides go further and advise keeping such peptides away from air altogether.
Cold, dark and dry
- Cold slows every reaction.
- Dark removes the trigger for photo-oxidation.
- Dry removes a reactant and keeps the molecules still.
One more point connects storage to paperwork. A certificate of analysis describes the material on the day it was tested. How the material has been stored since then decides how well that description still holds; see How to read a certificate of analysis.