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Does aspartimide formation dominate for liraglutide held at 40 °C?

Asked 23 Apr 2024Modified 2.0 years agoViewed 21k times
8

Conditions: aspartimide formation · liraglutide · 40 °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.

Why does this happen, and what would falsify the usual explanation?

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M4
askedmz_4113101k35823 Apr 2024
3Same question here after a warm delivery, so I am following this. – helena_vidmar 3 months ago
4Worth saying whether the vial has been opened, because that starts a different clock. – bac_or_bust 4 months ago
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5 Answers

Accepted answer first, then by votes
110

Accepted answer

At 40 °C the question is which route is fastest, not whether aspartimide formation happens — and the routes do not share an activation energy, so their ranking changes with temperature. 40 °C is 35 kelvin above the 5 °C middle of a 2–8 °C refrigerator. The ten-degree rule of thumb — degradation rate roughly doubling per 10 K — makes that about 11 times the refrigerated rate, which is an order-of-magnitude statement and not a shelf life. That multiplier is an average over every route at once, which is exactly why it cannot tell you which one wins. A cyclic imide at Asp, eighteen daltons lighter, which then reopens to a mixture including the iso-aspartyl form — same formula as the parent, different molecule, and invisible to a mass-only method. So the way to answer it for your vial is to pick the method that sees aspartimide formation specifically and run it against a control held cold, rather than to infer a mechanism from a purity number that averages all of them.

Start with the sequence, because which pathways are available depends on which residues are present.

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.

Reported and extrapolated stability by condition

StateConditionUsable windowBasis
Lyophilised solid−20 °C, sealed, dry24–36 monthsSupplier guidance
Lyophilised solid2–8 °C, sealed12–24 monthsSupplier guidance
Lyophilised solid25 °C, sealed4–8 weeksExtrapolated (Arrhenius)
Lyophilised solid40 °C, sealed1–2 weeksExtrapolated
Solution, preserved2–8 °C28 daysUSP microbiological convention
Solution, preserved25 °C3–7 daysExtrapolated
Solution, unpreserved2–8 °C24 hoursUSP microbiological convention

Windows for the solid state are chemical; windows for solution are microbiological and usually shorter than the chemical limit.

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.

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

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

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P9
answered · acceptedplate_count_9k78k2487 Jul 2024
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99

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.

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.

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

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

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

edited 19 Jul 2024 by dmitri_savchuk — fixed an arithmetic slip in the third paragraph

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DS
answereddmitri_savchuk27k3826 Jun 2024
Is there a reason to prefer minus eighty here, or is minus twenty genuinely enough? – zeynep_arslan 4 months ago
8Confirming that opening a cold vial in a humid room is a genuinely bad idea. – Dr_Nadia_Farsi 3 months ago
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49

Aggregation is a physical process and is the one most often caused by handling rather than by time.

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.

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

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

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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EL
answeredesben_lykke84k1584 Jun 2024
39

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

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.

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

At dilute concentrations, suspect adsorption before you suspect chemistry.

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DB
answeredDr_Ingrid_Baumgartner73k5815 Jun 2024
Does the same reasoning apply to material already in solution, or is that a different curve? – bac_or_bust 5 months ago
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32

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

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 kinetics for asparagine in peptides are well characterised and strongly sequence-dependent: the residue following the asparagine dominates the rate, with glycine and serine at the n+1 position accelerating it by an order of magnitude relative to bulkier residues. That is why two peptides in the same buffer at the same temperature can have quite different shelf lives.

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

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TV
answeredten_mg_vial31k13810 Aug 2024
Any published figure for how much a collapsed cake actually retains? – Dr_Yusuf_Adeyemi 9 months ago
Worth adding that residual moisture predicts this better than any printed date. – nkem_obiora 18 days ago
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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.