How lyophilised peptides are stored: what stability studies show

Freeze-drying is the standard way to supply research peptides because a dry solid is far more stable than the same peptide in solution. “More stable” is not the same as “stable indefinitely”, though. Chemical reactions continue in the dry state, only more slowly, and how slowly depends on temperature, on how much water the powder holds, on light and oxygen, and on what else is in the vial. This article summarises what published stability studies describe about the lyophilised state, and what those findings mean for how a laboratory keeps vials on the shelf. It deals only with the dry powder.

Much of the evidence comes from pharmaceutical work on peptides and proteins, where long, controlled stability studies are done. Where a finding was made on a specific molecule, such as insulin or an antibody, it is named, because it may not transfer to another sequence.

What does lyophilisation do to a peptide?

Lyophilisation removes water in stages. The solution is frozen, so that most of the water turns to ice. In primary drying, the ice is removed by sublimation under vacuum. In secondary drying, the temperature is raised to drive off water that is still bound to the solid rather than frozen. What remains is a porous “cake” that keeps roughly the shape of the frozen fill (Wang, 2000).

Freezing and drying are themselves stresses. For proteins they can disturb structure, and formulations often contain excipients chosen to protect the molecule during the process and in storage (Wang, 2000). Two roles are usually distinguished. Fillers such as mannitol give the cake mechanical structure and often crystallise. Stabilisers such as sucrose or trehalose stay amorphous, forming a glass in which the peptide or protein is embedded. Reviews describe two main explanations for how amorphous sugars protect proteins in the solid state: they replace some of the hydrogen bonds that water would otherwise form, and they immobilise the molecule in a rigid glass where reactions are slow (Chang and Pikal, 2009). Many research peptides are freeze-dried with no excipient at all, in which case the peptide and its counter-ions form the solid on their own.

Secondary drying never removes every molecule of water. The water left behind, the residual moisture, is usually reported as a percentage of the cake’s mass and measured by methods such as Karl Fischer titration. It is one of the most important variables in the stability literature, as the sections below show.

A further concept recurs throughout: the glass transition temperature (Tg). An amorphous solid below its Tg is a rigid glass; above it, the solid becomes rubbery and molecular mobility rises sharply. Water acts as a plasticiser, meaning it lowers Tg. In a freeze-dried antibody formulation, Tg fell from about 80 °C at 1% residual moisture to about 25 °C at 8% (Breen et al., 2001). A cake that is comfortably glassy when dry can therefore be close to its glass transition at room temperature once it has taken up water.

Which degradation pathways continue in the dry state?

A review of solid-state chemical stability lists the main reactions reported for peptides and proteins in lyophilised form: deamidation, peptide-bond cleavage, oxidation, the Maillard reaction (with reducing sugars), beta-elimination, and dimerisation or aggregation (Lai and Topp, 1999). A later review of protein pharmaceuticals covers the same chemical pathways alongside physical instability and discusses stabilisation in the dried state (Manning et al., 2010).

Deamidation of asparagine and glutamine. The side-chain amide of asparagine (Asn), and more slowly glutamine (Gln), can be removed to form aspartate or glutamate, often through a cyclic intermediate. The local sequence matters, which is why rates differ widely between peptides (Manning et al., 2010). The solid state does not stop this chemistry. In lyophilised insulin powders held at 35 °C, the main route was an intramolecular attack at the C-terminal asparagine of the A chain, forming a cyclic anhydride that then gave either the deamidated product or a covalent dimer. That intermediate formed readily even in the glassy state, apparently because that short segment kept some flexibility while the molecule as a whole was immobile (Strickley and Anderson, 1996).

Aspartate reactions and peptide-bond cleavage. Aspartate (Asp) residues can form a cyclic imide that leads to isomerisation or to cleavage of the backbone. A model hexapeptide containing an Asp-Gly sequence (Val-Tyr-Pro-Asp-Gly-Ala) degraded in lyophilised formulations, and a factorial study of that peptide is one of the clearest examples of how storage variables interact (Oliyai et al., 1994); it is described in the moisture section below.

Oxidation. Methionine can be oxidised to its sulfoxide, cysteine thiols can form disulfides, and tryptophan, histidine and tyrosine side chains are also susceptible (Manning et al., 2010). Oxidation is a common degradation pathway in solid as well as liquid formulations. It often proceeds by autoxidation, a chain reaction that can be started by trace metal ions or peroxides and that needs molecular oxygen to propagate (Hovorka and Schöneich, 2001). Reactive impurities in excipients, such as peroxides, are another recognised source of degradation (Waterman and Adami, 2005).

Aggregation. In the dry state, aggregation can be covalent or non-covalent. When lyophilised insulin was exposed to raised temperature and humidity, it formed both kinds of aggregate. The covalent route was traced to beta-elimination of an intact disulfide bridge, followed by thiol-catalysed disulfide exchange between molecules (Costantino et al., 1994).

Diketopiperazine formation. A peptide can cyclise at its N-terminus, cutting off the first two residues as a diketopiperazine. Kinetic work on model peptides with proline in the second position found that the rate depended on how much of the N-terminal amino group was un-ionised and on which residue preceded proline (Goolcharran and Borchardt, 1998). Those measurements were made in solution. Quantitative data for diketopiperazine formation in lyophilised peptides are sparser, so the relevance for a given dry peptide is best judged from that peptide’s own stability data.

How does temperature affect solid-state stability?

Lower temperature slows chemical reactions, which is why lyophilised peptides are commonly stored refrigerated or frozen. The relationship is not always the simple one predicted from a single activation energy, however. In the freeze-dried antibody study, vials with 1% to 8% residual moisture were stored between 5 °C and 50 °C for 6 or 12 months. All formulations were stable at 5 °C. In the glassy state, both the chemical and the physical degradation pathways followed Arrhenius kinetics. Above Tg only Asp isomerisation still did, and degradation was better fitted by the Williams-Landel-Ferry model used for rubbery materials (Breen et al., 2001).

Reviews of shelf-life prediction from stress data make the same point: degradation mechanisms can change at high temperature, so rates measured under stress do not always extrapolate cleanly to storage conditions (Waterman and Adami, 2005). For amorphous solids, the glass transition is an obvious place where such a change can occur.

For a laboratory, the practical consequences are a consistent storage temperature well below the cake’s likely Tg and few warm excursions. Each trip from cold storage to the bench also risks bringing in moisture, described below.

How do moisture and humidity affect a lyophilised peptide?

Water is the variable most often singled out in the solid-state literature (Lai and Topp, 1999). Several studies on specific molecules illustrate why:

• In lyophilised insulin at 35 °C, the rate of degradation rose with the water content of the powder at hydration levels well below the glass transition, and approached the rate in solution near the water content needed to induce a glass transition. The authors described the decomposition as a water-induced solid-state reaction (Strickley and Anderson, 1996).
• In a separate study of lyophilised insulin, the extent of aggregation correlated directly with the water taken up by the powder, as read from its water sorption isotherm (Costantino et al., 1994).
• For the lyophilised Asp hexapeptide, a factorial design tested starting pH, temperature, residual moisture and filler excipient (amorphous versus crystalline). Residual moisture, temperature and especially the type of filler excipient had statistically significant effects on degradation; the pH of the starting solution did not. The importance of moisture and temperature also depended on which excipient was present (Oliyai et al., 1994).

Drier is not automatically better for every pathway. In the antibody study, higher moisture lowered chemical stability whether the cake was glassy or rubbery, but aggregation behaved differently: intermediate-moisture vials were more stable to aggregation than the driest ones when stored below Tg (Breen et al., 2001). The general lesson is that the residual moisture set at manufacture is a designed property of the material, and uncontrolled moisture uptake after manufacture is the risk to manage.

Amorphous lyophilised solids are typically hygroscopic: the porous cake has a large surface area and readily takes up water from humid air. ICH Q5C, which covers well-characterised proteins and polypeptides, notes that humidity testing can usually be omitted when a product is shown to be in a humidity-protecting container (ICH Q5C). In laboratory terms, the closed vial is the moisture barrier, and most moisture uptake happens when it is open.

Why should a cold vial reach room temperature before it is opened?

Air holds less water vapour the colder it is. When a vial comes out of a refrigerator or freezer, the glass and the powder inside are below the dew point of the room air. If the vial is opened while still cold, moist room air enters, cools on contact with the cold surfaces and the powder, and water can condense on them or be absorbed directly by the hygroscopic cake. Because the studies above link water content to the rate of deamidation, aggregation and other reactions, a habit that adds water each time a vial is opened works against the purpose of freeze-drying.

The usual laboratory handling point follows from this: leave the vial sealed until it has reached room temperature, ideally inside a desiccator or a closed container with desiccant so that condensation forms on the outside rather than on the contents. Open it for as short a time as possible, close it promptly, and return it to cold storage. Where a batch is supplied in several vials, opening one at a time keeps the others sealed and unexposed.

What do desiccation and inert-gas headspace add?

Desiccation. Storing sealed vials in a desiccator, or a sealed box with desiccant, lowers the humidity around them. It does not meaningfully dry the cake further, but it limits moisture passing an imperfect closure and gives the vial a dry place to warm up.

Inert-gas headspace. Freeze-dried vials are often stoppered under vacuum or after the drying chamber has been backfilled with dry nitrogen, which leaves little oxygen and little water vapour in the headspace. Because autoxidation needs molecular oxygen to propagate (Hovorka and Schöneich, 2001), limiting headspace oxygen is relevant for sequences containing methionine, cysteine or tryptophan. Once a vial has been opened, room air replaces that headspace. Some laboratories re-blanket opened vials with dry nitrogen or argon before closing them; the published evidence on how much this extends stability for a particular peptide is limited, so it is best seen as a precaution rather than a measured safeguard.

How do light and oxygen affect a lyophilised peptide?

Light drives a distinct set of reactions. The residues that undergo primary photo-oxidation in proteins are tryptophan, tyrosine, phenylalanine and cysteine or cystine. A review of photodegradation in protein biologics describes these pathways and the methods used to limit photodamage, while noting that specific data on photodegradation of biopharmaceuticals are limited (Kerwin and Remmele, 2007). Keeping vials in their carton or another opaque container limits exposure on the shelf. ICH Q1A(R2) treats photostability as an integral part of stress testing of a drug substance, alongside temperature, humidity and oxidation.

For oxygen, the points made above apply: autoxidation needs it and occurs in solid formulations as well as in solution (Hovorka and Schöneich, 2001).

What do accelerated studies and ICH guidelines say about extrapolation?

Formal stability testing is defined by ICH guidelines. ICH Q1A(R2) sets standard storage conditions:

Intended storage	Long-term condition	Accelerated condition
Room temperature	25 °C / 60% RH (or 30 °C / 65% RH)	40 °C / 75% RH, 6 months
Refrigerator	5 °C ± 3 °C	25 °C / 60% RH, 6 months
Freezer	−20 °C ± 5 °C	none defined

For substances intended for freezer storage there is no accelerated condition, and the re-test period should be based on real-time data at the long-term condition. A single batch is instead tested at a higher temperature (for example 5 °C or 25 °C) to address short excursions, such as during shipping or handling (ICH Q1A(R2)). Stress testing goes further, using temperatures in 10 °C steps above 40 °C, high humidity, oxidation and light, to reveal degradation pathways rather than to set a re-test period.

ICH Q1A(R2) allows only limited extrapolation of real-time data beyond the period observed, and says it must be justified by what is known about the degradation mechanism, accelerated data, the fit of the mathematical model and supporting data. ICH Q1E sets out when this is acceptable. Where there is no significant change at the accelerated condition, a proposed period can be up to twice the period covered by long-term data, but not more than 12 months beyond it; both guidelines stress that extrapolation assumes the same change pattern will continue, and Q1E adds that any extrapolated period should be confirmed by further long-term data as they become available (ICH Q1E).

For proteins and polypeptides, ICH Q5C goes further. It states that expiry dating should be based on real-time, real-temperature data; accelerated and stress studies are encouraged as supporting information, but the conditions used for small molecules may not suit these products and should be chosen case by case (ICH Q5C).

The scientific literature explains the caution. Arrhenius extrapolation assumes that the same mechanism operates at every temperature, yet mechanisms can change at higher temperatures (Waterman and Adami, 2005), and in amorphous solids the behaviour above and below Tg can differ (Breen et al., 2001). A forecast built on 40 °C data for a cake whose Tg is near that temperature, especially a cake that has absorbed water, may not describe what happens at −20 °C.

What does this mean for reading a storage statement?

A storage instruction on a vial or datasheet is shorthand for a body of stability data, or for its absence. Useful questions when reading one:

• Which temperature and how long, and was that period measured in real time or estimated from short high-temperature studies?
• Does the certificate report residual water, and is the vial supplied sealed under vacuum or inert gas?
• Does the sequence contain residues with known solid-state vulnerabilities, such as methionine, cysteine, tryptophan, asparagine (especially before glycine), aspartate before glycine or proline, or proline in the second position?
• Is the material supplied with an amorphous stabiliser, a crystalline filler, or no excipient at all?

For most laboratories the handling points that follow from the literature are modest: keep vials sealed, cold, dry and dark; let them reach room temperature before opening; keep opening time short; and regard any shelf-life figure as only as strong as the real-time data behind it. Our guide to reading a Certificate of Analysis covers where residual water and counter-ion content appear on the batch documentation.