Lyophilisation

Volatile ones disappear with the water and leave the protein or peptide behind: ammonium bicarbonate, ammonium acetate, dilute acetic acid, formic acid, plain water. These are the clean cases, and they are what peptide and oligonucleotide work uses.

Everything non-volatile stays in the cake. Freeze-drying 1 ml of PBS leaves about 9 mg of salt, and if you then redissolve in 200 µl you have a five-fold concentrated buffer. That is fine if you plan for it and a problem if you do not.

Sodium phosphate is the one to avoid outright. On freezing, disodium phosphate crystallises out preferentially and the pH of the remaining liquid falls by two to three units, down towards pH 4, before the sample is dry. Many proteins do not survive that. Potassium phosphate shifts the other way and much less. Histidine, citrate and Tris are more forgiving, though Tris changes pKa strongly with temperature and some amine buffers partly sublime.

If the buffer is not compatible, the fix is a desalting step into a volatile buffer before drying, which we can do in the same order.

Peptides and small stable proteins usually come through unharmed. Larger proteins, multi-subunit assemblies and anything that depends on a specific hydration shell often do not, and the damage happens in two separate places: at the freezing step, where ice formation concentrates everything left in the liquid, and at the drying step, where the water bound to the protein surface is removed.

A lyoprotectant addresses the second. Sucrose or trehalose at 1-10% (w/v) replaces surface water with hydrogen bonds of its own and holds the protein in an amorphous glass. As a rule of thumb the sugar should be at least equal to the protein by mass, and a several-fold excess is more usual. Mannitol is a bulking agent that gives a solid cake but crystallises, so it does not protect on its own and is used with sucrose rather than instead of it. A non-ionic surfactant at 0.01-0.05% suppresses aggregation at the ice-water interface.

For a protein that has never been lyophilised before, drying a small test aliquot and checking activity and aggregation after reconstitution costs one day and saves the batch.

From a few hundred microlitres in an Eppendorf tube to several hundred millilitres in a flask. What matters more than the total is the fill depth: below one to two centimetres, because the sublimation front has to travel up through the already-dried layer and the drying time goes up steeply with thickness.

Wide, shallow containers dry faster than tall narrow ones. For a round-bottom flask, shell freezing — rotating the flask in a cold bath so the liquid freezes as a thin layer on the wall — turns an impossible geometry into a good one.

Tubes must be left open with a pierced cap or a membrane, not sealed. And they should not be filled to the brim: as the pressure drops, a sample that is not fully frozen can foam and climb out.

Overnight for a small aqueous sample in a thin layer; two to four days for a large volume, a thick fill or a formulation with a lot of sugar.

Three stages set the total. Freezing, which is quick but whose rate influences ice crystal size and therefore everything afterwards. Primary drying, where the ice sublimes — this is the long part, and it has to run at a temperature below the collapse temperature of the frozen solution, which for a sucrose formulation is around -32 °C and for trehalose around -30 °C. Secondary drying, where the water still bound to the solid is desorbed at a higher shelf temperature, typically several hours and aimed at a residual moisture of 1-3% or below.

Trying to shorten primary drying by raising the temperature is what produces collapsed cakes, so the schedule depends on the formulation rather than on the queue.

Add the volume of water or buffer you want, let it stand a few minutes without shaking, then swirl gently. Vortexing a protein solution generates foam and an air-water interface, which is where reconstituted proteins aggregate.

Redissolving in the original volume of water regenerates the original solution, salts included, if the buffer was non-volatile. Redissolving in a smaller volume concentrates everything, which is a legitimate way to concentrate a protein but changes the buffer at the same time.

Peptides dried from volatile buffers sometimes resist water and need a small amount of DMSO, or dilute acetic acid for basic peptides and dilute ammonium hydroxide or bicarbonate for acidic ones, before dilution into the working buffer.

Check what you get back: a quick A280 and a DLS measurement after reconstitution tell you whether the material survived, and take fifteen minutes.

Sealed and dry. A lyophilised cake reabsorbs water from the air within minutes of opening the tube, and moisture is what drives most of the slow degradation in the solid state. Close under vacuum or under nitrogen, seal with parafilm, add desiccant for long storage.

Stable peptides and small proteins keep for months at 4 °C and longer at -20 °C. Anything fragile should still go to -20 or -80 °C. Let the tube warm to room temperature before opening, otherwise condensation forms on the cold powder.

Shipping is the practical advantage: a dried sample travels at ambient temperature without dry ice, which changes what is possible for international shipments. Whether that is a good idea for your particular protein depends on whether it survived drying in the first place, which is the test to do before committing to the shipment.

Collapse is the classic failure. If the product temperature rises above the collapse temperature during primary drying, the amorphous matrix softens and the structure slumps into a sticky glassy residue instead of a light cake. It redissolves badly, retains more water and protects the protein less.

Loss of activity without any visible sign is the other one, and it is why an activity or aggregation check after reconstitution belongs in the plan rather than being an afterthought.

Smaller problems: a sample that was not fully frozen boils and foams as the vacuum is applied, and can end up on the ceiling of the chamber; a tube filled too high loses material the same way; a cake that dries too aggressively at the surface forms a skin and traps moisture underneath; oxidation-sensitive proteins degrade over months in the dry state if the tube is sealed under air.

Tell us what the sample is worth. An irreplaceable preparation is dried conservatively with a long primary drying step, and that decision is made before the run rather than after.

Often not, and the question is worth asking before the sample is committed.

For storage of a protein solution, flash-freezing small aliquots in liquid nitrogen and keeping them at -80 °C is gentler, faster and reversible. Lyophilisation earns its place when you need ambient-temperature shipping, when you need to remove a volatile buffer or solvent completely, when the final application requires a defined dry mass, or when the material has to sit at room temperature for a long time.

For removing solvent from a small peptide sample, a vacuum concentrator is quicker and adequate, though it heats the sample and offers no protection to anything fragile.

If the goal is simply to concentrate a dilute protein, centrifugal ultrafiltration is usually the better tool, since drying concentrates the salts along with the protein.

Peptides and synthetic oligonucleotides, purified proteins and protein complexes, buffer and media components, sugars and polysaccharides, and formulation trials where several excipient compositions are dried side by side and compared afterwards.

Detergent-containing samples are difficult. Most detergents do not dry to a solid and leave an oily residue that never fully redissolves, and a protein dried out of detergent rarely comes back in a defined micellar state.

Samples containing glycerol cannot be freeze-dried at all — glycerol does not sublime and does not freeze into a solid at the temperatures involved, so you end up with a syrup. Glycerol has to be removed by desalting first.

Live cells and viable material are outside what we do; that needs a validated protocol with specific cryoprotectants and is a different kind of work.

The sample, its composition — buffer, salt, any glycerol, detergent or sugar already present — the total volume, and what you intend to do with the dried material afterwards.

That last point changes the protocol more than anything else. Drying for long-term storage, drying to remove a volatile buffer before mass spectrometry, and drying to produce a defined mass for weighing are three different jobs with three different endpoints.

Say whether the sample can be frozen at -80 °C or whether it needs slower freezing, whether any activity assay exists for checking it afterwards, and whether you want us to add a lyoprotectant or to dry it as it is. If the material is irreplaceable, send a small test aliquot first — that is the single piece of advice that prevents the most losses.