Peptide stability: temperature, moisture and light

A peptide stored in a freezer is not chemically frozen in time. It continues to change, slowly or quickly, depending on its sequence, its physical state and its surroundings. This article describes the main ways peptides degrade, the storage conditions that accelerate each one, and why the research literature supports general principles rather than a single set of rules for every peptide.

It is written for laboratory work. It gives no instructions for storing products intended for personal or clinical use. Reconstitution and solution preparation are covered separately in our article on lyophilised peptides.

Two kinds of instability

Degradation is conventionally divided into chemical instability, in which covalent bonds are made or broken, and physical instability, in which the molecule's structure or state changes without a chemical reaction. The chemical pathways include proteolysis, deamidation, oxidation, racemisation and β-elimination. The physical pathways include aggregation, precipitation, denaturation and adsorption to surfaces^[1]. The two interact. A chemical change can make a peptide more likely to aggregate, and aggregation can expose residues to further reactions^[2].

Chemical pathways

Hydrolysis and aspartic acid

Peptide bonds can be broken by water, and some are far more vulnerable than others. Bonds next to aspartic acid are a well-studied example. In a model hexapeptide containing aspartic acid, the dominant reaction at strongly acidic pH was cleavage of the bond after the aspartic acid residue. At pH 4 to 5, cleavage and conversion to an isomer through a cyclic intermediate occurred together. Above pH 6, isomerisation was the only product observed^[3]. The same residue can therefore follow different degradation routes depending on pH.

Deamidation

Asparagine residues can lose their side-chain amide group through a cyclic succinimide intermediate. This produces a mixture of aspartic acid and isoaspartic acid forms, with some racemisation. In one model peptide, the asparagine residue deamidated with a half-life of only 1.4 days at 37 °C and pH 7.4. Replacing the neighbouring glycine with a bulkier residue slowed the reaction 33- to 50-fold^[4]. Sequence context matters as much as the residue itself.

N-terminal cyclisation

A glutamine at the N-terminus can cyclise spontaneously to pyroglutamic acid. In model peptides, this reaction depended strongly on temperature and buffer composition, and less on pH^[5].

Oxidation

Methionine, cysteine, histidine, tryptophan and tyrosine are the residues most susceptible to oxidation. Oxidation can be triggered by contaminating oxidants, catalysed by trace metal ions or induced by light. It is influenced by pH, temperature and buffer composition. Antioxidants are not a universal remedy. They can inhibit oxidation driven by oxidants in the system, but in metal-catalysed oxidation adding an antioxidant may accelerate the reaction^[6].

Light

Light is often overlooked. In proteins, the residues that undergo primary photo-oxidation are tryptophan, tyrosine, phenylalanine and cysteine or cystine. Photodegradation can alter structure at several levels. Data on biopharmaceuticals themselves are limited^[7]. For peptides containing these residues, protection from light during handling and storage is a reasonable precaution. How much protection is needed cannot be predicted from general principles.

Physical pathways

• Aggregation. Peptides can self-associate into amorphous aggregates or ordered fibrils. The factors involved include sequence, concentration, pH, net charge, excipients, surfaces, temperature, agitation and freeze-drying^[8].
• Interfaces. Contact with air–water interfaces, container materials and other surfaces is a major trigger of aggregation in solution^[9].
• Adsorption. Peptides can bind to the walls of containers, and recovery varies considerably between glass, plastic and treated surfaces^[10]. For some cationic peptides, 90% or more was lost from solution at typical experimental concentrations^[11]. During storage, adsorption reduces the amount of peptide available even though none has degraded chemically.

Solid state versus solution

Most degradation reactions need water, or are much faster when water is present. This is why many peptides and proteins are stored cold or freeze-dried to achieve an acceptable shelf life^[12]. Freeze-drying does not abolish instability. Dried proteins can still have limited long-term stability^[13].

The solid state has its own variables. In a factorial study of the aspartic acid hexapeptide in freeze-dried formulations, residual moisture, temperature and especially the type of bulking agent significantly affected chemical stability. The pH of the original solution did not^[14]. The importance of moisture and temperature depended on which bulking agent was present. This is a useful example of interaction between formulation variables.

Moisture

Water acts both as a reactant and as a plasticiser that increases molecular mobility in a dried solid. In a lyophilised antibody formulation stored for up to a year, the glass transition temperature fell from 80 °C at 1% moisture to 25 °C at 8% moisture. Higher moisture reduced chemical stability whether the solid was in a glassy or rubbery state. Physical stability, by contrast, was not compromised by higher moisture if storage remained below the glass transition temperature, and it may even have improved^[15]. "Drier is better" is therefore true for some pathways and not for others.

Temperature

Lower temperatures generally slow chemical reactions, and in the glassy state degradation in the antibody study followed predictable temperature dependence^[15]. Temperature is not a simple dial, however. Near and above the glass transition temperature, degradation kinetics changed^[15]. This is one reason why stability at one temperature cannot always be extrapolated reliably to another.

Freezing and thawing

Freezing is not a neutral event. Low temperature, freeze-concentration of solutes and ice formation are the three main stresses. Freeze-concentration can also accelerate reactions, cause buffer components to crystallise and redistribute solutes^[16]. The choice of buffer matters. When sodium phosphate buffers freeze, crystallisation of the disodium salt can lower the pH by as much as three units^[17]. The rates of freezing and thawing also affect how much damage occurs, and recrystallisation during thawing adds further stress^[18]. Each freeze–thaw cycle repeats these stresses, which is why stock solutions are commonly divided into single-use portions.

Why there is no universal storage rule

Every factor above depends on the peptide. A sequence without methionine, cysteine or tryptophan is less exposed to oxidation. One with an asparagine–glycine pair is especially prone to deamidation. A hydrophobic or amyloid-prone sequence is more likely to aggregate. Formulation adds another layer: buffer, pH, excipients, counter-ions and container all change which pathway dominates.

For this reason, laboratory recommendations on peptide storage tend to emphasise characterisation and documentation rather than fixed rules. A consensus from clinical proteomics, for example, set out recommendations for characterising, storing and handling peptide standards used in mass spectrometry^[19]. Where stability matters for a result, it has to be measured, typically by repeated chromatographic analysis under the intended storage conditions.

Summary

Peptides degrade through chemical pathways, including hydrolysis, deamidation, cyclisation and oxidation, and through physical pathways, including aggregation and adsorption. Moisture, temperature, light, pH, freezing and container surfaces each accelerate some of these routes. Their effects depend on sequence and formulation, and they can interact. Freeze-drying and cold storage slow degradation but do not stop it. Because stability is specific to each peptide and formulation, it should be established by measurement rather than assumed from a general rule.