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Does dimerisation dominate for cagrilintide held at minus 80 °C?

Asked 1 Apr 2024Modified 2.2 years agoViewed 18k times
This question was closed as needing more focus.Closed 19 Apr 2024. Answers already posted are preserved; new answers are not accepted. Questions here should ask one identifiable thing.
5

What I am working with: dimerisation · cagrilintide · minus 80 °C.

The empirical answer seems settled. The explanation does not.

If the honest answer is that nobody knows, I would rather hear that than a plausible story.

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

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askeddmitri_savchuk27k381 Apr 2024
Same question here after a warm delivery, so I am following this. – bea_castellanos 10 months ago
Worth saying whether the vial has been opened, because that starts a different clock. – dmitri_savchuk 8 months ago
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4 Answers

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7

At minus 80 °C the question is which route is fastest, not whether dimerisation 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. Two chains join, usually through a disulfide, so the product is roughly twice the mass and shows up as a late peak — or as nothing, if it never comes off the column. So the way to answer it for your vial is to pick the method that sees dimerisation 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.

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.

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.

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

edited 13 May 2024 by Dr_Ingrid_Baumgartner — added the citation requested in comments

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answeredDr_Ingrid_Baumgartner73k5830 Apr 2024
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5

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.

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.

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

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

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answeredten_mg_vial31k13823 May 2024
3Same experience here, different supplier. – esben_lykke 10 months ago
4I would add a sentence about light, since tryptophan-containing sequences care. – k_szabo 41 days ago
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4

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

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.

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

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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answeredpriya_menon13k353 Jun 2024
Two lots stored differently, reassayed at a year — the difference was smaller than I expected. – Dr_Ravi_Selvarajah 4 months ago
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3

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

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.

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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HC
answeredhaze_check9.3k1611 May 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.