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Does oxidation dominate for liraglutide held at minus 80 °C?

Asked 13 May 2024Modified 2.0 years agoViewed 20k times
18

Concretely: oxidation · liraglutide · minus 80 °C.

This is one of those things that everyone repeats and nobody derives.

This matters practically, not just academically, because it changes what I would do next.

Is the standard explanation correct, and if so, what is the evidence for it?

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BU
askedbufferline4230k13813 May 2024
4Is there a printed date on the vial, and do you know what it was derived from? – nkem_obiora 9 months ago
3Voting to keep this open — it is more specific than it first looks. – marta_okonkwo 8 months ago
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5 Answers

Accepted answer first, then by votes
61

Accepted answer

At minus 80 °C the question is which route is fastest, not whether oxidation happens — and the routes do not share an activation energy, so their ranking changes with temperature. minus 80 °C is 85 kelvin below a refrigerator, and below the glass transition of a lyophilised cake the ten-degree rule of thumb stops applying at all — solid-state chemistry is not slow liquid chemistry, it is a different regime, and the failure modes that survive it are mechanical rather than chemical. That multiplier is an average over every route at once, which is exactly why it cannot tell you which one wins. 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. So the way to answer it for your vial is to pick the method that sees oxidation specifically and run it against a control held cold, rather than to infer a mechanism from a purity number that averages all of them.

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.

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.

Adsorption losses at low concentrations are quantified in formulation studies and are the reason carrier proteins are used in dilute preparations.

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

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answered · acceptedpierce_count24k3830 Jun 2024
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73

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

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.

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

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

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

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LW
answeredlinnea_wahlberg17k2722 Jul 2024
4Worth adding that residual moisture predicts this better than any printed date. – plate_count_9k 17 days ago
5Same experience here, different supplier. – kofi_mensah 2 months ago
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48

The honest answer is that most reported "degradation" is adsorption and dilution error rather than chemistry.

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.

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.

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

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

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NN
answerednine_point_nine60k1482 Aug 2024
28

This is answerable from the chemistry rather than from anecdote, which is unusual and welcome.

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.

Aggregation at air-liquid interfaces is established from surface-tension and particle-count studies and is the basis for anti-agitation handling guidance.

Nothing here is medical advice, and research-use compounds are not approved for human use.

At dilute concentrations, suspect adsorption before you suspect chemistry.

edited 30 Jul 2024 by forty_two_c — removed a claim I could not source

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answeredforty_two_c66k5811 Jul 2024
The doubling-per-ten-degrees rule is the part I did not know and now use constantly. – n_takahashi 44 days ago
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27

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

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.

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

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answeredforty_two_c66k588 Jun 2024

Your answer

Ask PeptideStack is a static archive. Posting is closed, but the norms are worth stating: answer the question that was asked, show your working, cite the trial or the certificate, and say plainly where the evidence runs out.

Not medical advice. Research-use-only compounds are not approved for human use.