Methionine oxidation (revision 19)

Methionine oxidation converts the thioether side chain of methionine to a sulfoxide, adding 16 daltons, and on further oxidation to a sulfone, adding 32. It is one of the most common oxidative degradation routes in peptides and is readily detected by mass spectrometry because the mass shift is large.^[1]

Oxidation is promoted by dissolved oxygen, by trace transition metals, by peroxide impurities in excipients, and by light. Unlike deamidation, which proceeds spontaneously in clean aqueous solution, oxidation generally requires an oxidant, and controlling the oxidant is therefore an effective control strategy.^[2]

The consequence for activity depends on where the methionine sits. A surface methionine remote from the binding region may be oxidised with little effect; one within the receptor-contact region can substantially reduce potency.^[1]

The sulfur of methionine is readily oxidised by two-electron oxidants such as hydrogen peroxide to the sulfoxide, a reaction that is fast and essentially irreversible under ordinary conditions. Further oxidation to the sulfone requires more forcing conditions and is less commonly observed in practice.^[1]

Metal-catalysed oxidation is the more insidious route. Trace iron or copper, in the presence of oxygen and a reducing agent, generates reactive species locally at metal-binding sites, so oxidation can be site-specific rather than uniform. Chelating agents in formulations exist largely to suppress this.

Peroxide contamination of excipients — polysorbates in particular are prone to peroxide formation on storage — is a recognised source in formulated products. For research peptides supplied as unformulated lyophilisates the more likely sources are the drying process, container headspace and light exposure.^[2]

The +16 Da shift is resolvable on essentially any mass spectrometer, so oxidation is one of the easier modifications to detect — a contrast with deamidation, where the 0.984 Da shift requires high resolution.^[2]

Chromatographically the sulfoxide is more polar than the parent and elutes earlier on reverse phase, usually with adequate resolution. It therefore appears as a distinct related substance on a competent method and is included in an itemised related-substances table; resolving it from the parent is part of what a specificity assessment demonstrates.^[3]

Control strategies are the ordinary ones: exclude oxygen from the headspace, protect from light, avoid metal contact, keep the material cold and dry. Antioxidants are used in formulated products but are not present in unformulated research material.^[1]

An itemised related-substances table showing a small oxidised species is an unremarkable finding and, as noted at Limit of detection, indicates a method sensitive enough to see it. Its absence from a table may mean it is absent from the material or that the method did not resolve it.^[2]

Where a certificate reports only a single aggregate figure for related substances, oxidation state cannot be determined from the document at all. Where it reports intact mass only, an oxidised fraction will not appear unless it is a large proportion, since the unoxidised species will still dominate the spectrum.

As with every degradation route, a determination made at release describes the lot at release. Oxidation continues during storage, more slowly in the dry solid than in solution but not at zero rate, which is one of the reasons a beyond-use date and a storage condition accompany a stability statement.^[4]

• Deamidation
• Peptide aggregation
• Mass spectrometry
• Beyond-use date
• Cold chain