In plain English
A peptide is a short chain of amino acids (the building blocks of proteins). Labs build peptides one amino acid at a time. No step is perfectly efficient, so a finished sample holds small amounts of the wrong molecules as well as the right one. These are called impurities. Some are almost the same as the target, such as a chain with one amino acid missing. Others are leftover chemicals from the making process, such as trifluoroacetate (TFA, a strong acid used in the process) [1, 2].
Labs find most impurities with HPLC (a method that separates the parts of a sample). They identify them with mass spectrometry (a method that weighs molecules). Neither method catches everything. Some impurities weigh exactly the same as the target, so they need special tests [3, 10].
Even a small impurity can change the result of an experiment [14].
The short version
- Every synthetic peptide sample contains some impurities.
- Most come from the making process or from slow breakdown over time.
- No single test finds all of them.
- The impurity limits set for medicines do not apply to research materials [1].
What counts as a peptide impurity?
European guidance on synthetic peptides describes purity as one of the most important critical quality attributes. It sorts impurities into two categories [1]:
- Peptide-related impurities contain structural elements of the target sequence. They come from the starting amino acids, from the synthesis itself, or from degradation during manufacture and storage.
- Non-peptide impurities include process reagents and their by-products, residual solvents, elemental impurities and potentially mutagenic impurities.
A 2014 review of peptide medicines organised related impurities in the same way. It also noted that these impurities can distort early functional studies and lead to erroneous conclusions [2].
The most common peptide impurities
| Impurity | How it forms | Typical mass difference from target | Ref |
|---|---|---|---|
| Deletion sequence | One or more amino acids missing because a coupling or deprotection step was incomplete | Minus the mass of the missing residue(s) | [1, 2] |
| Truncated sequence | Unreacted chains are deliberately capped (acetylated) during synthesis, leaving shortened, acetylated fragments | Much lower than the target | [1] |
| Insertion sequence | An amino acid is coupled twice in one step, for example from excess reagent or premature deprotection | Plus the mass of the extra residue | [1, 2] |
| Incomplete side-chain deprotection | A protecting group is not fully removed at cleavage, leaving a peptide-protecting-group adduct | Plus the protecting group’s mass | [1, 2] |
| Diastereomer (epimer) | One amino acid converts from the L- to the D-form during coupling, or a D-isomer comes from the starting material | None (same mass) | [1, 3] |
| Oxidation | Oxidation of susceptible side chains, especially methionine; can occur during acidic cleavage | +16 Da per oxygen atom | [1, 4] |
| Deamidation | Asparagine or glutamine side chains convert to the acid form | About +1 Da | [1, 2] |
| Aspartimide | Base-catalysed ring closure at aspartic acid during Fmoc removal; can reopen to β-aspartyl and D-aspartyl forms | −18 Da (ring), then 0 Da (rearranged forms) | [5, 6] |
| Diketopiperazine (DKP) | The first two residues cyclise and drop off, leaving a double-amino-acid deletion | Minus two residues | [2, 7] |
| Pyroglutamate | N-terminal glutamine or glutamic acid cyclises | Small loss (water or ammonia) | [1, 2] |
| Dimers, oligomers and aggregates | Covalent or non-covalent self-association during synthesis or storage | Multiples of the target mass, or non-covalent assemblies | [1] |
| Counter-ion (e.g. TFA) | Left from cleavage and HPLC purification | Not a peptide; measured by weight | [2, 8] |
| Unrelated peptides | Cross-contamination from other products, reported where GMP controls were inadequate | Unrelated mass | [2] |
Mass differences above follow from the change in atomic composition and are given as a guide only.
Impurities from starting materials
Protected amino acids are themselves made in bulk and can carry impurities. The wrong enantiomer, the wrong amino acid (for example isoleucine in place of leucine), dipeptides or β-alanine adducts can all be built into the growing chain [1]. This is why guidance expects starting-material specifications to control these impurities.
Impurities from synthesis and cleavage
Solid-phase synthesis repeats the same deprotection and coupling steps many times. A small inefficiency at each step leads to deletion sequences, while excess reagents can cause insertions [2]. Two side reactions are particularly well studied:
- Aspartimide formation. One review of Fmoc chemistry described it as the most serious side reaction in the method. It is caused by repeated exposure of aspartic-acid-containing sequences to base, and is a particular problem for long peptides [5]. Research on suppressing it remains active [6].
- Diketopiperazine formation. In a mechanistic study of tirzepatide synthesis, DKP formation and the resulting double-amino-acid deletion impurities occurred mainly during Fmoc removal and while certain proline-containing intermediates were held after coupling [7].
At the cleavage step, side-chain protecting groups are released as reactive cations. These can attack tryptophan, tyrosine or methionine side chains, so scavengers are added to the cleavage mixture [1]. For methionine-containing peptides, oxidation and S-alkylation during TFA cleavage are recognised side reactions [4]. See How Research Peptides Are Made for the synthesis steps.
Degradation impurities
Degradation can continue after manufacture. European guidance lists oxidation, hydrolysis, isomerisation, deamidation, cyclic imide (aspartimide) formation, diketopiperazine and pyroglutamate formation, β-elimination, disulfide exchange and acetylation as degradation pathways for synthetic peptides [1]. Which of these occur depends on the sequence. Storage conditions that limit them are covered in Lyophilised Peptides: Storage, Stability and Degradation.
How labs detect peptide impurities
| Technique | Best at detecting | Limitation |
|---|---|---|
| Reversed-phase HPLC (UV) | Separating and quantifying most related impurities | Co-eluting impurities can hide under one peak; impurities may not absorb UV like the target [1, 9] |
| LC-MS and LC-MS/MS | Identifying impurities by mass and fragment pattern | Isomers and epimers have the same mass as the target [10] |
| Chiral analysis | D-amino acid (enantiomeric) impurities | Usually needs hydrolysis and chiral separation; few validated methods [3] |
| Size-exclusion chromatography | Dimers, oligomers and aggregates | Applied where relevant [1] |
| Amino acid analysis | Composition; distinguishing leucine from isoleucine | Doesn’t show sequence order [11] |
| HPLC methods for TFA and acetic acid; GC for solvents | Counter-ions and residual solvents | Measured by weight, separately from purity [11] |
Because no single technique catches everything, European guidance recommends orthogonal methods. It specifically mentions mass spectrometry early in development to reduce the risk of co-elution [1]. Method details are in HPLC, LC-MS and UV-Vis: How Peptide Purity Is Actually Measured.
How much impurity is acceptable?
For medicines, the answer is set by regulators, and peptides have their own rules. Synthetic peptides are excluded from the scope of the general ICH guideline on impurities in new drug substances (ICH Q3A) [12]. Instead, the European Pharmacopoeia’s general monograph Substances for Pharmaceutical Use sets thresholds for peptide-related impurities [1]:
| Threshold | Level | Meaning |
|---|---|---|
| Reporting | above 0.1% | The impurity must be reported |
| Identification | above 0.5% | Its structure must be identified |
| Qualification | above 1.0% | Its safety must be justified with data |
Where co-eluting impurities are measured as one peak, the 1.0% qualification threshold applies to the combined peak unless otherwise justified [1]. These thresholds apply to medicines, not to research materials. They are still a useful benchmark for how closely impurities can be characterised.
Regulators also consider the immunogenicity risk of impurities in peptide medicines. For generic synthetic peptides, the US FDA’s abbreviated pathway asks sponsors to report any new impurities compared with the originator product, partly because new sequences could introduce T-cell epitopes [13].
Why impurities matter in research
A minor impurity can change an experimental result. In one study of synthetic amyloid-β42, a single deletion species (Aβ42Δ39) was identified as the main contaminant limiting both fibril formation and cytotoxicity in cell assays. It also prevented formation of a stable dimer seen in very pure samples. The authors stressed the importance of purity and provenance in biological research generally [14].
Counter-ions can also affect results. Trifluoroacetate has been reported to alter cell proliferation in culture at low concentrations. This is covered in Counter-Ions in Peptides: TFA vs Acetate vs HCl.
What this means in Australia
In Australia, impurities are addressed through the rules for medicines and for testing laboratories.
- Peptides are therapeutic goods. Peptide products are regulated as therapeutic goods under the Therapeutic Goods Act 1989. Unapproved peptide products are not on the ARTG (the Australian Register of Therapeutic Goods) and have not been assessed by the TGA for safety, quality or effectiveness [16].
- Medicines are made under GMP. Manufacturers of therapeutic goods generally need a licence, and good manufacturing practice (GMP) applies. The TGA sums up GMP as: quality cannot be tested into a batch; it must be built in at every stage of manufacture [18].
- Scheduling is compound by compound. Some research peptides are named in the Poisons Standard. For example, BPC-157 and TB-500 are Schedule 4 (prescription-only) and Appendix D, clause 5 (possessing them without authority is illegal) [15].
- Labels don’t change status. The TGA has stated that a “research use only” disclaimer does not make supply lawful. It does not change a product’s regulatory status, permit importation, or remove advertising or supply obligations [16]. The TGA also counts applying a label to a blank vial as a step in manufacture [17].
- Laboratory accreditation. In Australia, NATA accredits testing laboratories to ISO/IEC 17025. The standard covers method validity, staff competence, traceability of measurements, equipment calibration, quality control and reporting of results [19]. When reading an impurity result, check whether the laboratory’s accreditation covers the test reported.
This is general information, not legal advice. Laws change; check the current Poisons Standard and TGA guidance.
Frequently asked questions
What are the most common impurities in synthetic peptides?
Deletion and insertion sequences, incompletely deprotected peptides, diastereomers, oxidised and deamidated forms, aspartimide-related products and aggregates. Residual counter-ions such as trifluoroacetate are also common [1, 2].
What is a deletion sequence?
A peptide missing one or more amino acids, caused by an incomplete coupling or deprotection step during synthesis. It usually appears as a separate HPLC peak with a lower mass [1].
Can mass spectrometry detect every impurity?
No. Diastereomers containing a D-amino acid have the same mass as the target peptide. They need chiral or high-resolution chromatographic methods [3, 10].
What impurity levels are allowed in peptide medicines?
Under the European Pharmacopoeia, peptide-related impurities are reported above 0.1%, identified above 0.5% and qualified above 1.0% [1]. These thresholds apply to medicines, not research materials.
Is TFA an impurity?
TFA is a non-peptide residue from cleavage and purification. It is usually present as the peptide’s counter-ion, and its content can be measured and limited [1, 8].
References
- European Medicines Agency. Guideline on the development and manufacture of synthetic peptides. EMA/CHMP/CVMP/QWP/367182/2025. Adopted 4 December 2025; effective 1 June 2026. ema.europa.eu. Accessed 11 October 2026.
- D’Hondt M, Bracke N, Taevernier L, et al. Related impurities in peptide medicines. J Pharm Biomed Anal. 2014;101:2–30. doi:10.1016/j.jpba.2014.06.012. PMID: 25044089
- Badgujar D, Paritala ST, Matre S, Sharma N. Enantiomeric purity of synthetic therapeutic peptides: a review. Chirality. 2024;36(3):e23652. doi:10.1002/chir.23652. PMID: 38448043
- Nandhini KP, Alhassan M, Veale CGL, Albericio F, de la Torre BG. Methionine-containing peptides: avoiding secondary reactions in the final global deprotection. ACS Omega. 2023;8(17):15631–15637. doi:10.1021/acsomega.3c01058. PMID: 37151509
- Behrendt R, White P, Offer J. Advances in Fmoc solid-phase peptide synthesis. J Pept Sci. 2016;22(1):4–27. doi:10.1002/psc.2836. PMID: 26785684
- Kong MJW, van den Braak TJHP, Neumann K. Aspartimide formation and its prevention in Fmoc chemistry solid phase peptide synthesis. ChemBioChem. 2025;26(18):e202500490. doi:10.1002/cbic.202500490. PMID: 40857621
- Wang J, Berglund MR, Braden T, et al. Mechanistic study of diketopiperazine formation during solid-phase peptide synthesis of tirzepatide. ACS Omega. 2022;7(50):46809–46824. doi:10.1021/acsomega.2c05915. PMID: 36570276
- Sikora K, Jaśkiewicz M, Neubauer D, Migoń D, Kamysz W. The role of counter-ions in peptides: an overview. Pharmaceuticals (Basel). 2020;13(12):442. doi:10.3390/ph13120442. PMID: 33287352
- Kumar Kuril A. The critical need for implementing RRF in the accurate assessment of impurities in peptide therapeutics. Anal Chem. 2025;97(24):12480–12485. doi:10.1021/acs.analchem.5c02149. PMID: 40499007
- Lian Z, Wang N, Tian Y, Huang L. Characterization of synthetic peptide therapeutics using liquid chromatography-mass spectrometry: challenges, solutions, pitfalls, and future perspectives. J Am Soc Mass Spectrom. 2021;32(8):1852–1860. doi:10.1021/jasms.0c00479. PMID: 34110145
- McCarthy D, Han Y, Carrick K, et al. Reference standards to support quality of synthetic peptide therapeutics. Pharm Res. 2023;40(6):1317–1328. doi:10.1007/s11095-023-03493-1. PMID: 36949371
- International Council for Harmonisation. ICH Q3A(R2): Impurities in new drug substances. 2006. database.ich.org. Accessed 11 October 2026.
- De Groot AS, Roberts BJ, Mattei A, Lelias S, Boyle C, Martin WD. Immunogenicity risk assessment of synthetic peptide drugs and their impurities. Drug Discov Today. 2023;28(10):103714. doi:10.1016/j.drudis.2023.103714. PMID: 37467878
- Adams DJ, Nemkov TG, Mayer JP, Old WM, Stowell MHB. Identification of the primary peptide contaminant that inhibits fibrillation and toxicity in synthetic amyloid-β42. PLoS One. 2017;12(8):e0182804. doi:10.1371/journal.pone.0182804. PMID: 28792968
- Therapeutic Goods (Poisons Standard—October 2026) Instrument 2026 (F2026L01327). Federal Register of Legislation. legislation.gov.au. Accessed 11 October 2026.
- Therapeutic Goods Administration. Understanding your responsibilities when importing, compounding and supplying unapproved peptide products. Safety advisory, 13 April 2026. tga.gov.au. Accessed 11 October 2026.
- Therapeutic Goods Administration. Unapproved peptide product promoters and suppliers are put on notice. Media release, 20 July 2026. tga.gov.au. Accessed 11 October 2026.
- Therapeutic Goods Administration. Good manufacturing practice (GMP); and Manufacturing. tga.gov.au; tga.gov.au. Accessed 11 October 2026.
- National Association of Testing Authorities, Australia (NATA). Laboratory accreditation: ISO/IEC 17025. nata.com.au. Accessed 11 October 2026.
Important information and disclaimer
General information only. This article is published for general educational and informational purposes. It summarises published scientific literature and public regulatory information as at the “last reviewed” date shown above. It is not medical, health, pharmaceutical, veterinary or legal advice, and it must not be relied on as a substitute for advice from a qualified professional about your circumstances.
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Regulatory status. Many peptides are scheduled substances under the Poisons Standard. Some are prescription-only (Schedule 4), and some may not be possessed without authority (Appendix D). The TGA has stated that a “research use only” label does not make supply lawful, change a product’s regulatory status, or remove advertising or supply obligations. State and territory laws may also apply. You are responsible for knowing and complying with all laws that apply to you. Regulatory information in this article may change after the review date.
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