Peptide impurity types: deletion, truncation, oxidation and other related substances

Two families: made during synthesis, or formed afterwards

The EMA guideline on synthetic peptides sorts related substances by where they come from: impurities carried in from the protected amino acid starting materials, impurities formed during synthesis and cleavage, and degradation products formed during manufacture or storage, whose levels may rise over time. [1] The distinction matters for a buyer. Process impurities are fixed when the lot is made; degradation products can grow between the certificate date and the day you use the material.

Non-peptide impurities, such as process reagents, residual solvents, elemental impurities and nitrosamines, are a separate category with their own tests. [1] This guide covers the peptide-related ones: molecules that look like the target to an HPLC detector and often differ from it by a single atom or residue.

Process-related impurities

Deletion sequences are missing one or more residues because a coupling or deprotection step did not go to completion. Manufacturers limit them by repeating couplings and acetylating unreacted chain ends; that capping produces acetylated truncated sequences instead. [1] A deletion shows up in mass spectrometry as the target mass minus one residue mass: -57.021 Da for glycine, -113.084 Da for leucine or isoleucine, -128.095 Da for lysine. [6]

Insertion sequences carry one extra copy of a residue. The EMA lists three causes: free amino acid present in the protected building block, premature deprotection during a long coupling, and excess building block not washed away before the next deprotection. [1] The mass is the target plus one residue mass.

Starting-material impurities can carry the wrong amino acid into the chain. The EMA names incorrect enantiomers or diastereomers, beta-alanine residues, dipeptides and swaps such as isoleucine in place of leucine. [1] Leucine and isoleucine have identical residue masses (113.08406 Da), so a swap is invisible to an intact-mass check. [6] Stereoisomers formed by epimerisation during coupling also have exactly the target mass. [1]

During cleavage, reactive cations released from protecting groups can attach to tryptophan, tyrosine or methionine side chains, and protecting groups that were not fully removed leave heavier variants. [1]

Degradation-related impurities

The EMA lists the pathways that can create degradation products: oxidation, hydrolysis, isomerisation, deamidation, cyclic imide (aspartimide) formation, diketopiperazine and pyroglutamic acid formation, beta-elimination, condensation, disulfide cleavage or exchange, and acetylation. It specifically mentions oxidation of cysteine and methionine. [1] Bachem notes that peptides containing Cys, Met or Trp are prone to oxidation and that Asn, Gln, Met, Cys and Trp shorten shelf life. [7]

High molecular weight impurities, such as dimers, oligomers and aggregates, form by covalent or non-covalent linkage. The EMA says a specification line that only says "high molecular weight impurities" is not acceptable for medicines; the species should be named, and size-exclusion chromatography is one technique for them. [1]

Mass shift lookup table

Use this table when a certificate or a mass spectrum notes a peak near the expected mass. Shifts are monoisotopic and per event; two oxidations add about 32 Da. [2] [3] [4] [5] [6] A shift alone is a hypothesis, not an identification: several causes share a mass, and the table flags the common collisions.

Where the 0.1%, 0.5% and 1.0% figures come from

Synthetic peptides are outside ICH Q3A, the general impurity guideline for new drug substances. The EMA guideline instead cites the European Pharmacopoeia general monograph Substances for Pharmaceutical Use: peptide-related impurities should be reported above 0.1%, identified above 0.5% and qualified above 1.0%. Methods should be able to meet the 0.1% reporting threshold, and when impurities co-elute as one peak the 1.0% qualification threshold applies to the combined peak unless justified otherwise. [1]

Those are medicine rules, and a research-grade certificate is not required to meet them. They are still a useful yardstick. A certificate that lists a single main-peak figure and nothing else tells you nothing about any individual impurity. A certificate that lists every peak above 0.1% and names those above 0.5% gives you something you can compare between lots. If your experiment is sensitive to a specific variant, such as an oxidised form, ask for that variant to be reported by name.

A fictional certificate, decoded

Invented example: a 12-residue peptide containing one methionine, one asparagine and one glycine, with a stated monoisotopic mass of 1400.00 Da. The certificate reports HPLC purity 97.8% and lists three minor peaks with mass notes: 0.9% at +16.0 Da, 0.6% at +1.0 Da, 0.4% at -57.0 Da.

Read with the table: +16.0 Da fits methionine oxidation, a degradation product that can grow in storage. +1.0 Da fits deamidation of the asparagine, but the same shift would appear if a C-terminal amide were a free acid, so the note alone cannot tell them apart. -57.0 Da fits a glycine deletion, a process impurity fixed at manufacture. What the certificate cannot show is a diastereomer or a Leu/Ile swap co-eluting under the 97.8% main peak, because both have the target mass. Under the Ph. Eur. scheme two of these peaks would need to be identified, as they are above 0.5%. [1]

• Note each impurity peak with its percentage, retention time and mass shift.
• Mark which ones are degradation-type (oxidation, deamidation) and may change with storage age.
• Ask whether the main peak was checked for co-eluting isomers.
• Compare the same peaks across lots rather than only the main-peak figure.