Deamidation and isoaspartate in peptides: detection and prevention

How does deamidation happen?

Deamidation converts asparagine to aspartic acid or isoaspartic acid (and, more slowly, glutamine to glutamic acid). It is one of the commonest chemical changes in stored peptides and proteins, and it is easy to miss: the mass changes by less than 1 Da.

At neutral and basic pH, the backbone nitrogen of the residue after asparagine attacks the asparagine side chain, forming a five-membered succinimide ring and releasing ammonia. The ring then opens to give a mixture of aspartate and isoaspartate, typically with isoaspartate predominating. Isoaspartate inserts an extra carbon into the backbone, which can change structure even though the composition hardly changes.

Source: Deamidation and isoaspartate formation in proteins, Cell Mol Life Sci 2003 (PubMed 12943218)

Which sequences deamidate fastest?

• Asn-Gly: fastest, because glycine's small side chain leaves the backbone nitrogen free to attack.
• Asn-Ser and Asn-His: also relatively fast.
• Gln: much slower than Asn under the same conditions.
• Higher pH, higher temperature and phosphate buffers speed the reaction; flexible regions deamidate faster than structured ones.

How is deamidation detected?

• Mass: +0.984 Da per deamidation; resolvable by high-resolution MS but easily confused with the 13C isotope peak at low resolution.
• Chromatography: deamidated and isoaspartate forms often elute as close-running peaks near the main peak.
• Enzymatic: protein isoaspartyl methyltransferase assays detect isoaspartate specifically.
• Reviews of peptide-medicine impurities list deamidation products among the main related substances.

Source: Related impurities in peptide medicines, J Pharm Biomed Anal 2014 (PubMed 25044089)

How can a lab slow deamidation?

• Keep peptides lyophilised until needed; the solid state is far slower.
• Prepare solutions at mildly acidic pH where the method allows (around pH 4–5 minimises succinimide formation for many sequences).
• Store solutions cold and use within the period your own stability check supports.
• Record the lot number and preparation date so a degraded stock can be traced.

How does Sable document lots?

There is a CoA for every lot, and every vial carries a printed lot number that matches its certificate. A CoA is emailed within 2 hours of a request.

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