How research peptides are made and tested

Building the chain

In solid-phase synthesis the first amino acid is anchored to a resin and further residues are added one at a time, each protected so that only the intended group reacts. After every coupling, protecting groups are removed and the next residue is added.

Errors compound with length: a small percentage of failed couplings at each step means longer peptides carry more truncated and deletion impurities.

Cleavage and purification

The finished chain is cleaved from the resin with a strong acid, commonly trifluoroacetic acid (TFA), which also strips side-chain protecting groups. The crude product is then purified by preparative HPLC, which separates the target from its impurities.

The TFA used in that process is why many peptides are supplied as trifluoroacetate salts, and part of why vial weight exceeds peptide weight.

Freeze-drying

Purified fractions are pooled and lyophilised. Freeze-drying removes water at low temperature and yields a stable powder, but it leaves some residual moisture and hygroscopic behaviour.

Quality control

Not every batch is tested for everything. A CoA should state which tests were actually performed.

• Analytical HPLC for purity.
• Mass spectrometry for identity.
• Amino acid analysis or nitrogen content for net peptide content.
• Karl Fischer titration for water content.
• Endotoxin and sterility tests for cell-based or sensitive applications.