What Is a Lyophilised Peptide?

A lyophilised peptide is a peptide that has been freeze-dried: frozen, then held under vacuum so that the ice sublimes straight to water vapour, and finally dried further to strip off water bound in the solid. What remains is a dry, porous solid known as a cake, which for most peptides is white to off-white. Peptides are supplied this way because the dry solid, sealed in a vial with little residual water, keeps far better than the same peptide with water present.

Lyophilised is the British spelling and lyophilized the American one; lyophilisation and freeze-drying are the same process. This guide explains how the process works, where it sits in peptide manufacture, what the cake is, and how the vial is closed.

How freeze-drying works

Freeze-drying removes water without ever letting it pass through the liquid state during drying. It has three stages, and each removes a different kind of water.

The triple point of water lies at 0.01 °C and 611.657 Pa, about 6 mbar. Below that pressure ice cannot melt as it warms; it can only sublime. Primary drying is usually the longest stage, and in the review by Tchessalov and colleagues the critical first step in designing it is choosing the target product temperature.

That temperature has a ceiling. The freeze-concentrated glass softens above a characteristic temperature, the collapse temperature, and if the drying layer warms beyond it, the pore structure left by the ice can flow and close up. The consequences can include a shrunken or collapsed cake and a higher residual water content, because water escapes more slowly from a solid that has lost its open structure. Freezing matters too: the size of the ice crystals formed at this stage decides the size of the pores left after sublimation, and so how easily vapour escapes during primary drying.

Where lyophilisation fits in peptide manufacture

A synthetic peptide is usually assembled by solid-phase synthesis, cleaved from its resin in trifluoroacetic acid (TFA) and purified by preparative reversed-phase HPLC in water–acetonitrile mixtures containing an acid. The pooled fractions of pure peptide are then freeze-dried. The process sequence is described in solid-phase peptide synthesis explained.

Freeze-drying at this point does more than take away the water. Acetonitrile and excess volatile acid leave with it, and Sikora and colleagues note that the usual mobile-phase acids, phosphoric acid apart, can be removed by lyophilisation. What cannot leave is the portion of acid held as a counter-ion by the peptide's basic groups, so a peptide with basic sites is isolated as a salt, most often the trifluoroacetate. That counter-ion and the residual water are why the mass of a lyophilised solid is larger than the mass of peptide it contains; see peptide purity vs net peptide content.

A lyophilised peptide reaches its vial in one of two ways. A measured volume of solution can be dispensed into each vial and freeze-dried there, which gives a cake shaped by the vial; or peptide freeze-dried in bulk can be weighed into vials as a powder. Either way, the product in the vial is a dry solid.

The lyophilised cake

The cake is the solid skeleton left behind when the ice sublimes. Each ice crystal leaves a pore, so the cake is highly porous and light for its volume, and the peptide in it is usually amorphous rather than crystalline. That open, glassy structure is what lets vapour escape during drying, and also what lets the solid take up water vapour quickly if it is later exposed to air.

Its appearance varies more than its composition does:

Tchessalov and colleagues note that a less-than-elegant cake does not necessarily mean the material is out of specification. Appearance is a first check, not a measure of composition: identity comes from mass spectrometry, and purity from HPLC, as explained in how to read a peptide certificate of analysis.

Colour follows the chemistry. Most peptides have no group that absorbs visible light, so their cakes are white to off-white. A metal complex can be coloured: the copper(II) complex GHK-Cu is blue, for reasons explained in what is GHK-Cu?.

Residual water

Secondary drying lowers the water content but never takes it to zero. Freeze-drying cycles are designed to a residual water target, and in model formulations Tchessalov and colleagues work with targets such as 0.2%, 0.5% and 1% by mass. The figure for a particular peptide depends on its sequence, its counter-ions and the drying cycle, so it is measured rather than assumed.

Residual water is measured by Karl Fischer titration, the same method a certificate of analysis cites for water content. It matters for two reasons. It is part of the gross mass, so it lowers the share of that mass which is peptide. And it affects stability: Lai and Topp list moisture content, with temperature and the physical state of the solid, among the factors that govern solid-state degradation, and water acts as a plasticiser that gives the molecules in an amorphous solid more mobility.

Lyophilised peptides are often hygroscopic. Bachem notes that short peptides and arginine-rich peptides can be very hygroscopic, which is why the vial stays sealed and cold vials are left to reach room temperature before they are opened.

Why peptides are supplied lyophilised

• Stability of the solid. Water is a reactant in hydrolysis and deamidation and a plasticiser for the solid, so removing it slows most degradation routes.
• Clean composition. Volatile residues from purification, such as acetonitrile and excess acid, leave during drying, so the solid is essentially the peptide salt and a little water.
• A solid that can be analysed and weighed. Identity, purity, water and counter-ion content can all be measured on a stable material.
• Robust in transit. A dry solid tolerates short periods at ambient temperature; Bachem notes that peptides may be shipped at room temperature.

The dry solid is stable, not inert, so it still needs cold, dry and dark storage. How to store lyophilised peptides covers the sealed vial in detail.

Vials, stoppers and crimp caps

A peptide freeze-dried in its vial is closed in stages that keep the dry solid away from room air:

1. During drying, a slotted elastomer stopper, often butyl rubber, sits partly inserted in the neck of the glass vial, so vapour can escape through the slots.
2. At the end of the cycle, the shelves of the freeze-dryer press every stopper fully home while the vials are still inside the chamber, commonly after the chamber has been filled with dry nitrogen to a set pressure. The vial is sealed before it ever meets humid air.
3. After unloading, an aluminium crimp cap is pressed around the neck of the vial and the rim of the stopper, holding the stopper in place.

The elastomer stopper is also called a septum. Together, the stopper and the crimp cap are the closure that the storage statement assumes: while they are intact, the solid, the gas above it and the vial form a closed system.

Certificate status is shown on every product page, certificates of analysis are published in the COA Library where available, and a batch certificate is available on request. How appearance and storage are documented is set out on the quality page, and lyophilisation, lyophilised cake, crimp cap and septum are defined in the glossary.