Stability is the concept describing how long a material keeps its identity, quantity and quality when stored under defined conditions. Where venom is concerned, the question becomes: how similar will the profile measured today be when the same sample is measured again in six months or two years? The answer is not a single number; it depends on the form of the sample, its container and the conditions it has been exposed to.
Storage equipment and practice in shipping were described in the article on the cold chain, and what drying achieves in the article on venom lyophilization. This article steps back and asks what changes at the molecular level.
By what routes does change occur?
The changes seen over time in proteins and peptides fall into two groups. In chemical changes the bonds in the molecule change, that is, a new molecule appears. In physical changes the molecule itself stays the same, but its folding, its solubility or its relationship with other molecules changes.
| Change | What happens? | Accelerating factor |
|---|---|---|
| Oxidation | Residues such as methionine, tryptophan and cysteine react with oxygen | Light, oxygen, trace metals |
| Deamidation | Asparagine and glutamine side chains lose their amide group and turn into a differently charged residue | High temperature, neutral and basic pH |
| Hydrolysis | The peptide chain breaks at certain bonds | The presence of water, extremes of pH |
| Enzymatic degradation | The venom's own enzymes break down other components | The presence of water, a warm environment |
| Aggregation | Molecules stick together and form larger structures | Temperature fluctuation, moisture, repeated freezing |
| Surface adsorption | Molecules remain on the container wall | Dilute solution, an unsuitable container |
The enzymatic degradation row in the table is a risk specific to venom. Crude venom carries active enzymes alongside the peptides, and as long as water is present these enzymes can carry on working. Chilling and drying the sample quickly after collection is the most direct way of stopping this internally driven degradation; it is also one reason why aqueous working solutions are not left standing for long.
Which route comes to the fore depends on the sequence itself. The 36-residue sequence of the α-KTx 3.6 peptide identified in the reference batch contains one methionine and one tryptophan, both residues prone to oxidation. The six cysteines in the same sequence, on the other hand, bond to one another and hold the molecule tightly folded, giving it marked robustness. How a sequence is read with this in mind is described in the article on amino acid sequences.
Temperature, light and moisture
Temperature
The rate of chemical reactions increases with temperature. As a rough approximation, every ten-degree rise in temperature is taken to roughly double the rate of many degradation reactions; the factor varies with the reaction, but it explains why low temperature matters so much. Fluctuation matters as much as the average temperature. In a sample that warms and cools, each cycle gives the molecules a fresh opportunity to aggregate. Freezing is not without effect either: as water turns to ice, the dissolved substances become concentrated in the remaining liquid, and the pH of that liquid can shift as some buffer salts precipitate.
Light
Ultraviolet and short-wavelength visible light cause residues with aromatic rings to absorb energy. The absorbed energy can initiate light-triggered oxidation in these residues, above all in tryptophan and tyrosine. A wait of a few minutes on a laboratory bench may cause no problem on its own; but weeks spent in a transparent container, by a window or on a continuously lit shelf leave a cumulative effect. Container options for protection against light were compared in the article on packaging.
Moisture
Water is either a direct participant in most degradation reactions or the medium that makes movement easier. In a dry powder the molecules are almost immobile within a glassy solid, and this immobility is the real source of the dry form's stability. As the powder takes up moisture, this solid softens and the temperature at which it leaves the glassy state falls. A material that was previously stable even at room temperature then begins to change faster at that same temperature. Caking and a change of colour can be the first visible signs of this process.
How is stability monitored?
How a material changes over time is learned not by prediction but by measuring the same sample again at defined intervals. In the pharmaceutical sector the framework for such studies is defined by international guidelines; the same logic can be used in a simpler form for research materials. A typical study consists of the following steps:
- A number of samples prepared from the same batch, in the same container, are set aside.
- The samples are placed under the intended storage condition and, for comparison, under a more demanding one.
- At the start and at predetermined time points, one sample is opened and measured.
- The results are compared with the initial values and with the acceptance criteria.
An accelerated study run under demanding conditions gives an impression quickly, but it does not replace behaviour under the real storage condition; a degradation route may come to the fore at high temperature and never be seen at low temperature.
The acceptance criteria are also set at the start of the study. Which change counts as acceptable depends on the intended use: for an application that looks only at total protein content, small shifts in the profile may be unimportant, while for work targeting a single peptide the same shift may be decisive.
On the measurement side, the methods that define the venom profile are at the same time the indicators of change. New peaks appearing in the chromatogram, or a fall in the areas of the main peaks, may point to chemical change; bands appearing in the high molecular weight region in electrophoresis to aggregation; an increase in the insoluble fraction or a fall in the protein value to physical loss. The starting point is the batch's first analysis. The initial measurement values for our reference batch are published on the analysis page, and the storage condition is given in the product documents.
The practical conclusion for the receiving laboratory is this: every opening, thawing and period of standing is added to the sample's history. Writing in the usage log how many times the container has been opened and how long it stayed out makes it easier to look for the cause if an unexpected result comes up later.
