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Peptide sequence impurities: deletion, insertion and truncation

Deletion, insertion and truncation variants differ from the target peptide by one or more residues. How they arise in synthesis, how labs detect them and where COA evidence stops.

  • Thistle BioLabs
  • 11 min read
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  • Updated

Research-use notice: this article is for legitimate laboratory and educational research only. It does not provide guidance for human use, administration, dosing or treatment.

Peptide sequence impurities are by-products whose amino acid sequence differs from the target: a deletion is missing one or more residues, an insertion has extra ones, and a truncation is a chain that stopped short. They arise mainly from incomplete or repeated coupling steps during solid-phase synthesis. Because they can closely resemble the target peptide, a convincing chromatogram and a plausible intact mass do not rule them out. Whether they are present depends on the sequence, the process and the batch.

This does not mean that every synthetic peptide contains every possible sequence impurity. It means that a result labelled simply “purity” should be read alongside the analytical method, the reference material and the specific variants the method was designed to detect.

What is a sequence-related peptide impurity?

The target sequence is the intended order of amino acid residues. A sequence-related impurity differs from that order while often retaining much of the target peptide’s chemistry. The European Medicines Agency (EMA) guideline on synthetic peptides describes the three main types and how they form during solid-phase peptide synthesis (SPPS).

Type How it arises Mass signature
Deletion One or more amino acids missing because a coupling or deprotection step was incomplete Lower by the mass of the missing residue or residues; an internal deletion leaves the rest of the chain in place
Insertion An amino acid coupled more than once during a single coupling step Higher by the mass of the extra residue, for example about 57 daltons (Da) for glycine
Truncation A chain that stopped growing, often because unreacted sites were deliberately capped by acetylation Shorter than the target, often carrying an acetyl cap

The EMA guideline notes that deletion sequences can be avoided by repeating the coupling step, or by acetylating unreacted sites, which is the capping that produces acetylated truncated sequences. It traces insertions to free amino acid present in the protected amino acid being coupled, premature deprotection during long coupling reactions, or incomplete removal of excess amino acid derivative before the next deprotection step. Side reactions add further related species. A 2014 review of impurities in peptide medicines by D’Hondt and colleagues lists deletion and insertion of amino acids among the impurities linked to solid-phase synthesis. The actual profile is sequence- and process-dependent, so a general list cannot establish what is present in a particular material or batch.

Why are closely related sequences difficult to separate?

A single-residue change may alter molecular mass, charge, hydrophobicity and conformation. It may also leave enough of the parent structure intact for the variant to behave similarly during chromatography.

This is why a single analytical result rarely answers every question:

  • Chromatography can separate components, but two species may co-elute or respond differently at the detector.
  • Intact-mass measurement can reveal a mass difference, but it may not locate the changed residue or distinguish every near-isobaric explanation.
  • Tandem mass spectrometry breaks the peptide at its peptide bonds into fragment series, known as b and y ions. As Steen and Mann explain in their 2004 review, the mass differences between successive fragments read out the sequence, which helps locate a missing or extra residue.
  • High-resolution mass spectrometry can distinguish small exact-mass differences that lower-resolution instruments may merge.
  • Nuclear magnetic resonance (NMR) can provide an orthogonal, atom-level structural fingerprint where enough material and a suitable method are available.

ICH Q2(R2), the International Council for Harmonisation (ICH) guideline on validating analytical procedures, calls this specificity or selectivity: the extent to which other substances interfere with measuring the analyte. Where one procedure does not discriminate well enough, it recommends combining two or more. For a broader comparison, see high-performance liquid chromatography (HPLC) vs mass spectrometry. The related guide 99% purity: what does that number actually tell you? explains why an area percentage is not a complete identity statement.

What recent analytical studies show

A 2026 study in Rapid Communications in Mass Spectrometry by Wu and colleagues compared one commercially available chemically synthesised teriparatide material with two recombinant pharmacopoeial reference standards, using liquid chromatography–high-resolution mass spectrometry. The researchers identified six amino acid deletion impurities only in the chemically synthesised material. This is evidence about the materials and methods examined in that study; it is not evidence that the same deletion profile applies to other peptides or suppliers.

A 2026 study in the Journal of Pharmaceutical and Biomedical Analysis by Liu and colleagues examined apparent +56 Da impurities in a 36-residue model peptide and traced them to four distinct origins. One was a +57 Da impurity from a double glycine insertion during chain assembly, which overlapped with a +56 Da tert-butylation product when viewed by low-resolution mass spectrometry. High-resolution measurements also showed that some of the remaining signal came from non-covalent adducts and solvent clusters formed during electrospray ionisation. The practical lesson is narrow but useful: a nominal mass shift is a clue, not a structural identification.

Another 2026 study in the same journal, by Mishra and colleagues, used natural-abundance NMR to compare bivalirudin, a 20-residue synthetic peptide, with two defined impurity standards, including [desGlu13]-bivalirudin, a synthesis deletion variant missing the glutamate at position 13. Two-dimensional heteronuclear amide maps distinguished the parent peptide from its process impurities. The work demonstrates an orthogonal structural approach; it does not make NMR a routine requirement for every research material.

A 2025 study in Separations by Bosc-Bierne and Weller examined a pool of 32 synthetic peptides by ultra-high-performance liquid chromatography with high-resolution mass spectrometry (UHPLC-HRMS). Using targeted extracted-ion searches for frequently occurring by-products, the authors found only very small traces of deletion peptides in the sample examined, alongside dimers, pyroglutamate and aspartimide formation, deamidation and methionine oxidation. They described their list as a guide rather than a comprehensive one. That shows both the value and the limitation of searching for predefined candidates: a targeted list can find what it includes, but it does not automatically prove that every unexpected species has been considered.

Evidence level: keep analytical detection separate from biological meaning

Computational evidence. Software can generate theoretical deletion, insertion and truncation libraries, predict masses and prioritise fragment ions. This is useful for search design. A predicted variant is not proof that the variant is present.

In vitro analytical evidence. The studies discussed here are laboratory measurements using chromatography, mass spectrometry or NMR. They support conclusions about the samples and analytical conditions tested.

Animal evidence. No animal study is required to show that a sequence variant can be separated or structurally characterised, and no animal outcome is relied upon in this article.

Clinical evidence. The cited analytical papers include pharmaceutical reference materials as study systems, but this article draws no clinical conclusion. Detecting an impurity does not, by itself, establish a human effect, safety threshold or therapeutic consequence.

How can laboratories investigate sequence variants?

A proportionate workflow begins with the research question rather than a universal checklist. Useful considerations include:

  1. Define the target sequence precisely. Include terminal modifications, counterion context and any expected non-standard residues.
  2. Review the method’s selectivity. Ask which known or plausible sequence variants were included during method development.
  3. Use accurate mass carefully. Compare observed and theoretical exact masses, charge states and isotope patterns rather than relying on a rounded shift alone.
  4. Localise the change. Where necessary, use fragment-ion evidence or an orthogonal structural method to identify the affected region.
  5. Check recovery and response. A low detector response or poor chromatographic recovery can make an impurity appear smaller than it is.
  6. Record uncertainty. “Unidentified related species” is more accurate than assigning a structure that the data do not resolve.

Sequence variants can also sit beside other changes. Read the related guides on deamidation and isoaspartate analysis and peptide oxidation for examples where a mass change still needs structural context.

What can a supplier-provided COA establish?

A supplier-provided certificate of analysis (COA) may report a chromatographic purity result, an identity test and named impurities where those tests were performed. Its evidential value depends on the method, sample, date, acceptance criteria and connection to the relevant material or batch.

A COA does not automatically establish that every possible deletion, insertion or truncation variant was resolved, identified or quantified. Nor does an identity result for the main peak prove the identity of every smaller peak.

When you read a report, check:

  • whether the document identifies the relevant material or batch;
  • which analytical technique produced each reported figure;
  • whether the test was designed to separate known sequence-related impurities;
  • whether unknown peaks were reported, and how they were quantified; and
  • whether the identity evidence applies to the main component only, or to individual related species.

For a fuller framework, see How to read a peptide COA and What a COA cannot tell you. Thistle shows a batch laboratory report on each product page, and you can look up a batch on our batch report lookup.

Regulatory guidance is context, not product certification

The EMA guideline on the development and manufacture of synthetic peptides became legally effective on 1 June 2026. It explicitly describes deletion, insertion and truncated sequences among the quality considerations for synthetic peptides. It also cites European Pharmacopoeia thresholds for peptide-related impurities: reported above 0.1%, identified above 0.5% and qualified above 1.0%.

The US Food and Drug Administration’s 2021 guidance on generic versions of certain synthetic peptides names insertion and deletion among the sequence changes that peptide-related impurities can carry. It recommends sensitive, high-resolution procedures such as UHPLC-HRMS, asks applicants to identify each peptide-related impurity at 0.10% or more of the drug substance, and sets 0.5% as the limit for any new impurity not found in the reference product.

Both documents apply to medicinal products. They do not certify research materials, individual suppliers or particular batches, but they provide authoritative terminology and show why sequence-related variants need method-specific consideration.

The practical conclusion

Sequence is not a decorative line on a specification sheet. It is part of molecular identity. Deletions, insertions and truncations may be close enough to the target peptide to challenge a single chromatographic or mass-based result, so reliable interpretation depends on method selectivity and, where needed, orthogonal evidence.

For research-material assessment, the careful position is simple: read reported purity and identity figures as answers to defined analytical questions, not as proof that every possible sequence variant has been excluded.

Peptide sequence impurity FAQs

What is a deletion sequence in peptide synthesis?

A deletion sequence is a by-product missing one or more amino acids, usually because a coupling or deprotection step did not go to completion. Its mass is lower than the target by the mass of the missing residues.

What is the difference between a deletion and a truncation?

A deletion is missing residues but continues to the end of the sequence. A truncation stopped growing early, often because unreacted chains were capped, so it is shorter than the target.

Can HPLC detect deletion sequences?

Sometimes. A deletion variant may separate from the target peptide, but close variants can co-elute. Liquid chromatography–mass spectrometry (LC-MS), ideally at high resolution, is normally needed to identify what a related peak is.

How are insertion impurities identified?

An insertion raises the mass by the extra residue’s mass, which accurate-mass MS can detect. Tandem MS then locates the extra residue. At low resolution, an insertion can be confused with another modification of similar mass.

Does a COA list sequence impurities?

Only if the method was designed to separate and identify them. Many reports give a single purity figure and an identity result for the main peak, which do not rule out minor sequence variants.

Sources

Published by Thistle BioLabs, a UK supplier of research materials. This article summarises external research, not studies conducted by Thistle. Educational information only; no medical advice or recommendation for personal use.

Educational information only. This journal does not recommend a substance for personal use or provide medical advice.