The small peaks on a chromatogram: synthesis-related impurities in peptides
Oct
Most research peptides are made by solid-phase peptide synthesis, which builds the chain one amino acid at a time on a resin bead. Any step that does not go to completion leaves a slightly different peptide behind, and those near relatives are the small peaks around the main peak on an HPLC trace.
Because they differ from the target by one residue, one protecting group or one stereocenter, they are the hardest impurities to separate and the ones a purity method most needs to resolve.
This guide explains how synthesis-related impurities form, how they differ from the target sequence and what a certificate needs to show for them to be accounted for. It complements our explainers on what a 99% purity number actually measures and why powder weight is not peptide weight.
Purification removes most of these impurities. The purity figure describes what is left.
How solid-phase synthesis builds a peptide
Merrifield introduced solid-phase peptide synthesis in 1963, anchoring the first amino acid to an insoluble resin so that reagents could be washed away after each step. The method is still the basis of most peptide manufacturing.
In the common Fmoc approach, every residue is added in a repeating cycle:
- Deprotection. The Fmoc group protecting the free end of the growing chain is removed.
- Coupling. The next amino acid, with its own Fmoc group and side-chain protection, is attached.
- Washing. Excess reagents are rinsed off before the next cycle starts.
- Cleavage. After the last residue, a strong acid, usually trifluoroacetic acid, releases the peptide from the resin and removes side-chain protecting groups.
Small inefficiencies compound. If every coupling were 99 percent complete, a 20-residue peptide would come off the resin with about 82 percent full-length chains, because 0.99 multiplied by itself 20 times is roughly 0.82. At 30 residues the figure falls to about 74 percent. The rest is a family of closely related sequences, which is why crude peptide is always purified.
The families of synthesis-related impurities
D’Hondt and colleagues reviewed the related impurities found in peptide medicines and grouped those that come from solid-phase synthesis. The table follows their classification.
| Impurity type | How it forms | How it differs from the target |
|---|---|---|
| Deletion sequence | Incomplete Fmoc deprotection or coupling, so a residue is skipped | One amino acid missing; lower mass |
| Insertion sequence | Excess amino acid reagent adds a residue twice | One amino acid extra; higher mass |
| Truncated sequence | Chain growth stops early, often by deliberate capping of unreacted ends | Shorter chain, missing the residues after the stop |
| Diastereomer | Racemization of an amino acid during activation or deprotection | One residue in the D form; same mass |
| Protection adduct | A side-chain protecting group not fully removed at cleavage | Extra mass equal to the protecting group |
| Side-chain modification | Unprotected side chains react with synthesis reagents, or oxidize | Modified residue; mass shift depends on the reaction |
| Dimers and oligomers | Chains joined to each other, for example through cysteine | Roughly double the mass or more |
The same review lists two further groups that are not sequence variants: residual counterions such as trifluoroacetate from synthesis or purification, and contamination by unrelated peptides, which the authors attribute to a lack of appropriate good manufacturing practice.
Why near relatives are hard to separate
RP-HPLC separates molecules mainly by hydrophobicity. Removing one residue from a chain of 20 changes the overall hydrophobicity only a little, so a deletion sequence often elutes very close to the target. A diastereomer has the same composition and can come off at almost the same time.
The purity figure counts only what the method separates and detects. A related impurity that co-elutes under the main peak adds to the main peak’s area and raises the purity number instead of lowering it. That is why method details and the chromatogram matter more than the headline figure.
A diastereomer formed by racemization has exactly the same mass as the target, so an LC-MS identity result cannot detect it. McCarthy and colleagues note that chiral and isobaric amino acids may need additional techniques to fully characterize a peptide reference standard. A chirality test or a method shown to separate the D-isomer is what answers the question.
What purification removes and what it leaves
Crude peptide is purified by preparative reversed-phase HPLC, and fractions that meet the purity target are pooled and freeze-dried. This removes most of the deletion, truncation and adduct sequences, with some trade-offs.
Closely eluting impurities
Impurities that elute right next to the main peak are the hardest to remove. Narrowing the collected fractions raises purity but lowers yield, so the final purity reflects a deliberate balance.
Counterion
Trifluoroacetic acid in the purification mobile phase means peptides are often recovered as TFA salts unless the counterion is exchanged afterward. It is not a sequence impurity, but it adds weight to the powder.
Residual solvents
Solvents from synthesis and purification, such as acetonitrile, can remain at low levels after drying. They are measured by gas chromatography where a certificate reports them.
After purification, the impurity profile can also change in storage. Oxidation and deamidation products form slowly over time, as covered in our guide to how peptides degrade.
How regulators treat peptide impurities
Peptides fall outside the general impurity guideline for small molecules. ICH Q3A(R2) excludes peptides from its scope, along with biological products, oligonucleotides and others, so peptide impurities are addressed through product-specific guidance.
The clearest benchmark is the FDA’s May 2021 guidance on generic synthetic versions of five peptides of recombinant origin: glucagon, liraglutide, nesiritide, teriparatide and teduglutide. It expects applicants to identify new peptide-related impurities, justify each one and keep each at no more than 0.5 percent of the drug substance.
These rules apply to approved drug products, not to research materials. They are useful here as a measure of how carefully a single related impurity is expected to be characterized when the stakes are highest.
What a certificate should show
A certificate does not need to name every impurity to be useful, but it should give enough to judge whether the related impurities were separated and counted.
- The chromatogram. It shows the main peak, the related peaks around it and whether they are resolved from each other.
- Method conditions. Column, gradient and wavelength determine which impurities the method can separate.
- LC-MS identity. The observed mass of the main peak confirms the target, and masses of minor peaks can point to deletions, insertions or adducts.
- Batch and date. Each result belongs to one lot tested at one time.
These gaps leave the impurity question open:
- A purity figure with no chromatogram
- A chromatogram cropped tightly around the main peak
- No column, gradient or wavelength stated
- An identity result with no expected and observed mass
- Identical chromatograms across different batches
- No batch or lot number tying the result to a vial
Every lot we supply has its batch documents in our certificate library. If you need more detail on a batch, contact the team with the lot number.
- Solid-phase synthesis adds one residue per cycle, and small inefficiencies compound with length
- Deletion, insertion, truncation, diastereomer and adduct sequences are the main synthesis impurities
- Near relatives elute close to the target, so separation decides what the purity figure counts
- Diastereomers have the same mass and need chiral methods to detect
- Purification removes most impurities but often leaves a TFA counterion
- A chromatogram with method details is what lets impurities be checked
Frequently asked questions
They are near relatives of the target sequence that form during solid-phase synthesis, such as deletion, insertion and truncated sequences, diastereomers from racemization, and chains that still carry a protecting group. They differ from the target by a small change in sequence, mass or stereochemistry.
A peptide that is missing one amino acid because a deprotection or coupling step did not go to completion in that cycle. It has a lower mass than the target and often elutes close to it in RP-HPLC.
Every residue adds a coupling and a deprotection step, and small inefficiencies multiply across the chain. A longer sequence produces a larger share of related impurities in the crude product, which makes purification harder.
No. Diastereomers formed by racemization have the same mass as the target, and some impurities co-elute with the main peak. Chiral methods and well-resolved chromatography are needed alongside mass spectrometry.
No. ICH Q3A(R2) excludes peptides from its scope. For approved drug products, peptide impurities are addressed through product-specific guidance, such as the FDA’s 2021 guidance on generic synthetic peptides.
- Merrifield RB, 1963. Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide. Journal of the American Chemical Society, 85(14), 2149 to 2154. DOI
- D’Hondt M, Bracke N, Taevernier L, et al., 2014. Related impurities in peptide medicines. Journal of Pharmaceutical and Biomedical Analysis, 101, 2 to 30. DOI
- McCarthy D, Han Y, Carrick K, et al., 2023. Reference Standards to Support Quality of Synthetic Peptide Therapeutics. Pharmaceutical Research, 40(6), 1317 to 1328. DOI
- ICH Q3A(R2) Impurities in New Drug Substances. ICH, October 2006. View source
- US Food and Drug Administration, 2021. ANDAs for Certain Highly Purified Synthetic Peptide Drug Products That Refer to Listed Drugs of rDNA Origin. Guidance for Industry. View source
This article is general educational information about laboratory analysis and material handling. It is not medical, legal or regulatory advice, and nothing in it describes or recommends the use of any compound in people or animals. Materials supplied by Imperial Biolab are for research use only: not for human or veterinary use, not for diagnostic use, and not for use as a food, drug, dietary supplement or cosmetic. Sale is restricted to adults aged 21 or over. See the research use disclaimer for details.