PEPTIDES & PROTEINS

Supporting capabilities: Structural elucidation, particulates, fibrils

Figure 3- Orthogonal techniques for aggregate detection & characterisation

understanding of stability under real formulation and handling conditions.

This article describes a pragmatic approach to assessing peptide stability under formulation-relevant conditions, with an emphasis on orthogonal analytical techniques and the frequently overlooked size continuum from soluble aggregates to sub-visible and visible particulates.

Why instability pathways cannot be viewed in isolation

Peptide chemists encounter various recurrent chemical issues: oxidation( particularly at Met and Trp), deamidation( Asn and Gln), hydrolysis, disulphide exchange and, depending on sequence and conditions, isomerisation or racemisation. Physical instability can manifest as reversible self-association, irreversible aggregation, adsorption to interfaces and structural transitions that may progress to fibrillation, precipitation and particulate formation.

In practice, degradation pathways are interdependent and can reinforce each other. Chemical changes can perturb local polarity and conformational

stability and, in some cases, increase the exposure of hydrophobic patches, shifting self-association equilibria and altering aggregation behaviour. Likewise, deamidation can change charge states and thereby influence colloidal stability and self-association.

Interfacial stress at air-liquid, solid-liquid, silicone oil-water or ice-liquid interfaces is a primary driver of aggregation and particle formation. Under conditions that may also generate reactive species, for example agitation or cavitation, or in the presence of reactive leachables or oxidants, interfacial stress can cooccur with, or exacerbate, chemical degradation, most notably oxidation.

These dependencies justify an orthogonal analytical strategy that measures chemical integrity and physical stability in parallel under the manufacturing, storage and handling stresses peptide products actually encounter.

This interdependence also explains why stability questions map directly onto development decisions. Programmes must demonstrate product integrity and comparability,

define drug substance( DS) and drug product( DP) stability, and set storage conditions and expiry dating with patient safety and compliance in mind, while ensuring that observed changes are interpreted holistically rather than as isolated assay readouts( Figure 1).

Orthogonality is practical. Because chemical and physical liabilities can influence one another and may emerge at different stages and under different stresses, stability assessments should combine orthogonal readouts that probe both domains under formulation-relevant conditions.

GLP-1 receptor agonists illustrate how molecule and formulation design choices shape stability questions. Lipidated analogues, such as liraglutide and semaglutide, use reversible albumin binding to extend exposure. Liraglutide also forms fatty acidmediated heptamers in solution, a reversible self-association behaviour that contributes to its depot-like absorption kinetics.

Such association equilibria can be sensitive to concentration and formulation conditions; orthogonal methods help distinguish reversible

MAR / APR 2026 SPECCHEMONLINE. COM

35