Peptide degradation pathways: oxidation, deamidation, hydrolysis and aggregation in research samples

Methionine, cysteine, histidine, tryptophan and tyrosine are the residues most susceptible to oxidation, because they react readily with reactive oxygen species. Oxidation can be driven by contaminating oxidants such as peroxides, catalyzed by transition metal ions, or induced by light, and it depends on pH, temperature and buffer composition (Li, Schöneich and Borchardt, 1995).

The two mechanisms behave differently. Oxidation by oxidants already in the solution can be suppressed with antioxidants or radical scavengers, but metal-catalyzed oxidation is site-specific and some antioxidants can speed it up; chelating agents are the alternative (Li et al., 1995). On a mass spectrum, methionine sulfoxide appears 15.995 Da above the parent for each oxygen added; two free cysteines forming a disulfide appear 2.016 Da lighter.

Asparagine deamidation is one of the fastest chemical changes in peptides. In the model hexapeptide VYPNGA at 37 °C and pH 7.4, Geiger and Clarke measured deamidation through a succinimide intermediate with a characteristic time of only 1.4 days; the succinimide then hydrolyzed to a mixture of normal aspartyl and isoaspartyl peptides and also racemized. Replacing the Gly after Asn with a bulky Leu or Pro slowed degradation 33- to 50-fold (Geiger and Clarke, 1987).

pH changes the route as well as the rate. In acidic solution Asn-containing hexapeptides deamidated by direct hydrolysis to the Asp peptide only, while at neutral or alkaline pH they went through the cyclic imide and gave both isoAsp and Asp products; the residue on the C-terminal side of Asn controlled the rate at neutral to alkaline pH (Patel and Borchardt, 1990). Robinson and Robinson measured deamidation rates for 306 Asn sequences in model peptides at pH 7.4 and 37 °C, which is the standard reference for sequence effects (2001).

Deamidation adds 0.984 Da, which high-resolution LC-MS can resolve. Isomerization of Asp to isoAsp adds nothing, so it is invisible to an intact-mass check and appears only as a new, closely eluting HPLC peak. This is one reason purity by HPLC and identity by LC-MS answer different questions.

Peptide bonds can hydrolyze, especially next to aspartic acid under acidic conditions, giving two fragments whose masses add up to the parent plus one water (18.011 Da) (Manning, Patel and Borchardt, 1989). For slowly deamidating sequences, the Asn side chain can also attack the backbone and cleave the chain (Patel and Borchardt, 1990).

An N-terminal glutamine or glutamic acid can cyclize to pyroglutamate. From Gln this loses ammonia (−17.027 Da); from Glu it loses water (−18.011 Da). Chelius and colleagues documented pyroglutamate formation from N-terminal Glu in antibodies (2006).

Diketopiperazine formation removes the first two residues as a cyclic dipeptide. A process study of tirzepatide synthesis traced diketopiperazine formation, and the resulting double-amino-acid deletion impurities, to intermediates with a penultimate proline, and showed that lower storage temperature markedly improved intermediate stability (Wang et al., 2022). These deletion impurities are a reason to look for masses below the parent on an LC-MS report.

Aggregation is a physical change: molecules associate into soluble oligomers or insoluble particles. It depends on concentration, pH relative to the isoelectric point, temperature, agitation and surfaces, and it can follow from chemical changes: metal-catalyzed oxidation induced aggregation and precipitation of human relaxin (Li et al., 1995b; Wang, 2005). It shows up as a falling main peak, turbidity, or species at multiples of the parent mass.

Lyophilization slows most of these reactions by removing water, but freezing and drying are stresses of their own, and solid material still has limited long-term stability (Wang, 2000). Residual moisture lets solid-state deamidation and oxidation continue, which is why vials should be warmed to room temperature before opening so that condensation does not form on the powder.

On an HPLC report, degradation appears as extra peaks and a lower area percent for the main peak. On an LC-MS report, compare the observed masses with the expected ones and with the shifts in the table above: a peak 16 Da heavy suggests oxidation, about 1 Da heavy suggests deamidation, and fragments suggest hydrolysis. Because a COA describes the lot on its test date, a report that is old relative to the material's storage history says less about the current state of the sample.