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Does oxidation of oral semaglutide at 30 °C show up as a loss of content or of purity?

Asked 18 Jun 2024Modified 22 months agoViewed 43k times
31

Concretely: oxidation · oral semaglutide · 30 °C.

I want to know whether this is a real physical effect or an artefact of how it is measured.

What prompted the question is an inconsistency between two sources I otherwise trust.

What is the causal chain, and where does it stop being established?

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BN
askedbirk_nordahl16k3818 Jun 2024
3Same question here after a warm delivery, so I am following this. – Dr_Otto_Lindqvist 3 months ago
2Worth saying whether the vial has been opened, because that starts a different clock. – kelvin_lam 43 days ago
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5 Answers

Accepted answer first, then by votes
104

Accepted answer

At 30 °C it can show up as either, and which one depends entirely on whether the product still elutes under the main peak. Purity is a ratio of areas, so a degradant only costs purity if the method resolves it. Content is a mass against a standard, so a degradant costs content whenever the parent is consumed — resolved or not. Met and Trp take up oxygen sixteen daltons at a time, and the oxidised species is more polar, so on a reversed-phase column it elutes ahead of the parent rather than behind it. That is why the two measurements are not interchangeable and why an unchanged purity figure after an excursion to 30 °C is weak evidence: the method may simply be integrating the degradant along with the parent and reporting the sum as one peak.

On the detail: aggregation is a physical process and is the one most often caused by handling rather than by time.

Light exposure matters for tryptophan-containing sequences and for anything with a chromophore. Amber vials and a closed box are free mitigations.

Aggregation is physical: peptides unfold at air-liquid interfaces and associate. Shaking maximises that interface, which is why swirling and shaking produce visibly different outcomes on the same vial.

Metal-catalysed oxidation of methionine is documented across peptide and protein formulations and is why chelators appear in some formulations.

Sequence determines which pathways apply, so general statements are general.

Swirl, never shake. Aggregation is a handling problem more than a time problem.

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M4
answered · acceptedmz_4113101k3582 Oct 2024
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93

Asparagine and glutamine are the deamidation risk, and methionine is the oxidation risk.

Hydrolysis cleaves the backbone, most readily at aspartate-proline and aspartate-glycine sequences, and is acid-catalysed. In a dry solid it barely proceeds at all.

Freeze-thaw cycling drives aggregation through concentration at the ice interface and pH shifts as buffer components crystallise out at different rates. Each cycle costs something.

A mass spectrum names the pathway. Plus one, plus sixteen, minus eighteen.

edited 22 Sept 2024 by kwn_analytical — fixed an arithmetic slip in the third paragraph

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KA
answeredkwn_analytical147k35821 Sept 2024
45

Answering this needs the physical state, since a dry powder is protected from most of these and a solution is protected from none.

A mass spectrum resolves most of this: minus eighteen is dehydration or succinimide, plus one is deamidation, plus sixteen is oxidation, and an unchanged mass with a shifted retention time is an isomer.

Deamidation converts asparagine or glutamine to the corresponding acid via a succinimide intermediate, adding one dalton. It is base-catalysed, accelerates above neutral pH and is the dominant aqueous pathway for many peptides.

Cold, dry, dark, still. Those four words cover most of the mitigation.

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DL
answeredDr_Otto_Lindqvist72k5830 Aug 2024
4Worth adding that residual moisture predicts this better than any printed date. – sian_llewellyn 2 months ago
3This should be in the site help pages rather than buried in an answer. – m_haraldsen 18 days ago
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36

The short version: water enables most of it, oxygen enables oxidation, surfaces enable adsorption, and agitation enables aggregation.

Oxidation targets methionine, cysteine and tryptophan, adding sixteen daltons per oxygen. It is catalysed by trace metals and promoted by dissolved oxygen and by light.

At dilute concentrations, suspect adsorption before you suspect chemistry.

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M4
answeredmz_4113101k35810 Sept 2024
5Small correction: it is the number of cycles rather than the freezer temperature that does the damage. – meniscus_film 2 months ago
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30

The relevant point is that a mass shift of plus one dalton is deamidation and plus sixteen is oxidation, so degradation is often visible in a mass spectrum if anyone looks.

Adsorption onto glass and plastic is significant at low concentrations — micrograms per millilitre — and negligible at milligrams per millilitre. It is the usual explanation for an apparent loss in a dilute preparation.

Deamidation via the succinimide intermediate is well characterised, with sequence-dependent rates highest for asparagine-glycine motifs.

Apparent loss in a dilute preparation is usually adsorption rather than degradation and is worth ruling out first.

Sequence decides which pathways are even available. Check the residues.

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KA
answeredkwn_analytical147k3588 Jul 2024
3Same experience here, different supplier. – tandem_gradient 7 months ago
2Is there a reason to prefer minus eighty here, or is minus twenty genuinely enough? – halvard_ness 5 months ago
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