Source of Peptide aggregation

{{Infobox concept | name = Peptide aggregation | subtitle = Degradation route | image = peptide-chain.svg | Reversible form = Self-association into defined oligomers | Irreversible form = Amyloid-like fibrils, amorphous particles | Detected by = Size-exclusion chromatography, light scattering, visual inspection }} '''Peptide aggregation''' is the association of peptide molecules with one another. It ranges from reversible self-association into defined oligomers, which may be a stabilising feature of a formulation, to irreversible formation of amyloid-like fibrils or amorphous particles, which is a degradation route.{{r|manning2010}} The distinction between the two is central and is frequently lost. [[Liraglutide|Liraglutide]] self-associates into heptamers in its formulation, and that association contributes to its duration of action; [[Amylin|human amylin]] forms fibrils, which is why it could not be developed as a medicine without engineering. Both are "aggregation" in a loose sense and they are not the same phenomenon.{{r|westermark2011}} Aggregation is driven by concentration, by pH near the isoelectric point, by hydrophobic surface exposure, by agitation at air–liquid interfaces, and by temperature. It is the degradation route most likely to be visible: a solution that has gone faintly hazy after [[Reconstitution of lyophilised peptides|reconstitution]] has changed.{{r|manning2010}} == Mechanisms == Aggregation proceeds through partially unfolded or conformationally exposed states rather than from the native state directly. Anything that increases the population of such states — heat, interfaces, shear, extremes of pH — increases the rate.{{r|manning2010}} Nucleation-dependent aggregation, characteristic of amyloid formation, shows a lag phase during which nuclei form, followed by rapid growth. The lag is why a solution can appear stable for a period and then change quickly, and why seeding with pre-formed aggregate abolishes the lag entirely — a relevant consideration when material is drawn repeatedly from one container. Interfacial aggregation is the mechanism most often encountered in handling. The air–liquid interface presents a hydrophobic surface at which peptides adsorb and unfold, so shaking a vial is substantially worse than swirling it. The same applies to repeated passage through a narrow needle.{{r|usp1503}} == Detection == | Method | Detects | Limitation | |---|---|---| | Visual inspection | Visible particles, haze | Insensitive to soluble oligomers | | Size-exclusion chromatography | Soluble aggregates | Dissociating conditions can hide reversible species | | Dynamic light scattering | Particle size distribution | Dominated by the largest particles present | | [[Reverse-phase HPLC]] | Little or nothing | Aggregates often dissociate on the column | The last row is the important one for reading certificates. A reverse-phase purity determination is run under denaturing conditions with organic solvent and acid; aggregates dissociate and are counted as monomer. A high purity figure by that method says nothing about aggregation state.{{r|usp1503}} Size-exclusion chromatography is the ordinary determination for aggregate content, and its result depends on the mobile phase: a dissociating buffer will report a cleaner profile than a native one on the same material. For an amylin analogue or an acylated incretin peptide, which one was used is a material question.{{r|manning2010}} == Practical consequences == Aggregation is the principal reason peptides are supplied [[Lyophilisation|lyophilised]] rather than in solution, and the principal reason a [[Beyond-use date|beyond-use date]] on a reconstituted solution is shorter than the shelf life of the powder.{{r|usp1503,ich_q1a}} Handling practices that reduce it are well established: dissolve by adding diluent down the vial wall rather than directly onto the cake, swirl rather than shake, avoid repeated freeze–thaw, and keep solutions cold. These follow from the mechanisms rather than from any particular guidance document. See [[Reconstitution of lyophilised peptides]]. For certificates, the point is one of method coverage. A specification for an aggregation-prone peptide that reports only reverse-phase purity omits the attribute most likely to fail for that chemistry. That is a statement about which questions the document answers, not about the material it describes.{{r|manning2010}} == References == {{reflist}} <ref name="manning2010">Manning MC, Chou DK, Murphy BM, Payne RW, Katayama DS. "Stability of protein pharmaceuticals: an update." ''Pharmaceutical Research'' 27(4):544–575 (2010). DOI:10.1007/s11095-009-0045-6. PMID 20143256.</ref> <ref name="westermark2011">Westermark P, Andersson A, Westermark GT. "Islet amyloid polypeptide, islet amyloid, and diabetes mellitus." ''Physiological Reviews'' 91(3):795–826 (2011). PMID 21742788.</ref> <ref name="usp1503">United States Pharmacopeia, General Chapter <1503>, ''Quality Attributes of Synthetic Peptide Drug Substances''.</ref> <ref name="ich_q1a">International Council for Harmonisation, ''Q1A(R2): Stability Testing of New Drug Substances and Products'' (2003).</ref> == See also == * [[Deamidation]] * [[Methionine oxidation]] * [[Lyophilisation]] * [[Reconstitution of lyophilised peptides]] * [[Amylin]] * [[Beyond-use date]] {{DEFAULTSORT:Peptide aggregation}} [[Category:Peptide degradation]] [[Category:Analytical science]] [[Category:Cold chain and stability]]