How research peptides are made: solid-phase synthesis and where COA impurities come from

Why the manufacturing route matters to a certificate reader

A certificate of analysis is a list of answers to questions about what could have gone wrong. You can only judge whether the right questions were asked if you know how the material was made. For short and mid-length catalog peptides, the usual route is solid-phase peptide synthesis (SPPS). Bachem lists SPPS for peptides under about 80 amino acids and liquid-phase synthesis for peptides under about 10, with fragment and ligation methods for longer chains. [2]

The European Medicines Agency guideline on synthetic peptides, which came into effect on 1 June 2026, describes SPPS as building the sequence on a solid support by repeated cycles of deprotection, washing, coupling and capping, followed by a final cleavage and deprotection step, purification and isolation. [1] That guideline is written for medicines, not research-grade catalog material, but it is the clearest official list of what each step can leave behind. This guide uses it as a map, not as a claim that any research supplier follows it.

The synthesis cycle, step by step

The chain is anchored at its C-terminal end to beads of polystyrene cross-linked with 1% divinylbenzene; Bachem names Wang and 2-chlorotrityl resins for peptides with a free C-terminal acid. The beads must swell in solvents such as DMF or NMP so reagents can reach the growing chain. [2]

Each cycle has four parts. First the temporary protecting group on the chain end (Fmoc in the more common strategy) is removed, Bachem says with piperidine. Then the resin is washed. Then the next protected amino acid is coupled. Then the resin is washed again. Synthesis runs from the C-terminus to the N-terminus. Because the chain stays on an insoluble bead, excess reagents are removed by washing rather than by isolating intermediates. Completion of coupling is checked with a colour test; the EMA guideline names the Kaiser test, based on ninhydrin reacting with free amines, with the chloranil and TNBS tests as alternatives. [1] [2]

When coupling stays incomplete, manufacturers commonly repeat the coupling and then acetylate (cap) any chain ends that still did not react, so they stop growing. The EMA guideline notes this capping is what produces acetylated truncated sequences. [1]

Why longer sequences come out dirtier

Bachem notes that by-products accumulate as chains get longer, which makes the crude product harder to purify by standard chromatography. [2] Neither Bachem nor the EMA gives a per-cycle yield, so here is purely illustrative arithmetic to show the shape of the problem. If every coupling were 99% complete and nothing were capped, about 90.4% of chains would be full length after 10 couplings, 74.0% after 30 and 60.5% after 50. At 99.5% per step the figures are 95.1%, 86.0% and 77.8%.

Those numbers describe crude material before purification, not the vial you receive. Their lesson is that a 30-residue crude product can contain a large family of near-identical failure sequences, each one residue short at a different position. Many differ from the target by a single amino acid and can sit close to the main peak on a chromatogram. That is why purification and the resolving power of the final HPLC method matter as much as the synthesis.

Cleavage, scavengers and the TFA question

In Fmoc chemistry, the side-chain protecting groups (Boc, tBu, OtBu, Trt, Pbf) are removed with trifluoroacetic acid (TFA), which also releases the peptide from the resin. [2] The EMA guideline explains that some protecting groups leave as reactive cations that can attach to sensitive side chains such as tryptophan, tyrosine and methionine, which is why scavengers are added to the cleavage mixture, and that incomplete removal of protecting groups is another source of impurities. [1]

TFA also stays with the product. Bachem states that peptides with basic groups (the N-terminus, Arg, Lys, His) form salts with TFA after Fmoc synthesis or with acetic acid, and that these counter-ions cannot be removed during purification. [3] The EMA guideline says acetate is the usual counter-ion for peptide medicines, that trifluoroacetate and chloride are possible, and that the counter-ion type should be defined and its amount controlled with a justified upper limit; its specification list includes a separate residual TFA test. [1] For a buyer, the practical point is that a research peptide described only as "99% pure" says nothing about whether it is a TFA salt, an acetate salt or something else, and the counter-ion changes how much peptide a weighed milligram holds.

Purification and freeze-drying

Bachem describes the standard purification as reverse-phase HPLC on C18-modified silica: polar contaminants elute first in water with 0.1% TFA, then acetonitrile (also with 0.1% TFA) is increased to separate the target, impurities are monitored at 210 to 220 nm, and fractions are checked by analytical HPLC, pooled and freeze-dried. [3] The analytical HPLC on your certificate is usually a close relative of this method, so impurities that co-elute in purification can also hide under the main peak in the purity test. The EMA guideline says that if one method cannot separate all peptide-related impurities, additional independent methods may be needed, and that control of diastereomers may require specific methods. [1]

The pooled fractions are lyophilised to a fine white powder. Bachem notes that complete water removal is not possible, and that net peptide content varies with the lyophilisation process, polarity, storage and humidity. [3] [4] The EMA guideline notes that precipitation, crystallisation and spray drying are alternative isolation methods. [1]

What a certificate should cover, and what it usually does not

Bachem lists HPLC purity, identity by mass spectrometry, amino acid analysis, peptide content, water by Karl Fischer titration, counter-ion content (acetic acid by HPLC or ion chromatography, residual TFA by ion chromatography) and residual solvents by gas chromatography among peptide QC tests, and mentions optical purity as an important parameter for amino acid derivatives and loaded resins. [4] The EMA specification list adds high molecular weight impurities, bacterial endotoxins and microbiological purity. [1]

Many research-grade certificates report two lines: HPLC purity and a mass check. That is enough to say the main peak has roughly the right mass and is a given share of the UV signal. It is not enough to say how much of the powder is peptide, what salt it is, or whether a diastereomer is hiding under the main peak. Use the table above to decide which missing lines matter for your work and ask for them before ordering.

• Ask which synthesis strategy was used (Fmoc or Boc) and whether the counter-ion is TFA, acetate or chloride.
• Ask for the HPLC method (column, gradient, wavelength) and the full chromatogram, not only the purity figure.
• Ask whether residual solvents and water were measured on this lot, and by which method.
• For long sequences, ask whether any impurity above 0.5% was identified and what it was.
• Record which lines were reported, which were requested and which were refused.