Physical degradation: aggregation and adsorption

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A peptide can lose activity without any covalent bond breaking: it is enough for its physical state to change.

Aggregation

Molecules associate with each other through their hydrophobic regions. Aggregates can be soluble and invisible or grow into particles and cloudiness. Some peptides (native human amylin is the classic example) form ordered amyloid fibrils. Aggregation:

• It reduces the amount of active peptide available.
• It can increase immunogenicity, because aggregates are more easily recognized as foreign.
• It is favored by high concentrations, pH near the isoelectric point, shaking and freeze-thaw cycles.

Mechanical stress and interfaces

Shaking a vial creates bubbles: every air-liquid interface is a surface where the peptide partially unfolds and aggregates. That is why the universal instruction to swirl gently and not shake has a precise physical basis.

Adsorption to surfaces

At low concentrations, an appreciable fraction of the peptide can stick to the glass or plastic of the vial and syringe. It is a real problem with very large dilutions and one of the reasons it is not advisable to prepare extremely dilute solutions.

Freeze-thaw cycles

On freezing, water crystallizes and the peptide becomes concentrated in the liquid channels left between the crystals, together with salts and buffer. That «cryoconcentration» and the associated pH changes favor aggregation. Each cycle adds damage: hence the recommendation to divide into aliquots and not refreeze what has been thawed.

Educational content for scientific purposes. It distinguishes preclinical from clinical evidence and is not medical advice, diagnosis or a recommendation for use. NeoPeptidos products are sold labelled for research use only (RUO).