Reference
How peptides are made
Almost every peptide sold is built one residue at a time on a plastic bead. Understanding that process explains something a purity figure never states outright: why a synthetic peptide is never entirely pure, and why longer ones are harder.
The problem
Making a peptide means joining amino acids in one exact order. That is harder than it sounds, because amino acids have reactive groups at both ends. Left to themselves in solution they will join to each other in whatever order they meet, producing a mixture rather than a sequence.
Every technique for making peptides is really a technique for controlling that — forcing one specific bond to form at one specific moment, and preventing every other bond that could form instead.
Solid-phase synthesis
The method that solved it was published in 1963 by Robert Bruce Merrifield, who received the Nobel Prize in Chemistry for it in 1984. It is still how nearly all synthetic peptides are made today.
The idea is to anchor the growing chain to something you can wash. The first amino acid is attached to an insoluble resin bead. Everything after that happens in a cycle:
- Deprotect. Remove the blocking group from the end of the chain, exposing it.
- Couple. Add the next amino acid, in large excess, so the reaction runs to completion.
- Wash. Rinse everything unreacted away. The chain stays put because the bead does.
Then repeat. A twenty-residue peptide is nineteen turns of that cycle. The genius of it is the washing step: because the product is physically tethered, excess reagents can be flushed out without purifying anything, and the whole process can be automated.
Protecting groups
Each incoming amino acid arrives with its own reactive sites blocked, so it can only join at the intended position. Two strategies dominate, named for the group used on the chain end: Fmoc and Boc. Fmoc is more common in modern work because it is removed under milder conditions.
Side chains carry their own protection, removed at the end. A sequence rich in reactive side chains needs more of this, which is one reason two peptides of identical length can differ considerably in how difficult they are to make.
Why purity is never 100%
Each coupling step is efficient but not perfect. If a step succeeds 99% of the time, then 1% of chains on the beads did not receive that residue — and they carry on through the remaining cycles as a chain that is one residue short. These are deletion sequences, and they are the characteristic impurity of solid-phase synthesis.
The arithmetic compounds, which is the part worth seeing written out:
| Chain length | Couplings | At 99% each | At 99.5% each |
|---|---|---|---|
| 5 residues | 4 | 96% | 98% |
| 10 residues | 9 | 91% | 96% |
| 15 residues | 14 | 87% | 93% |
| 20 residues | 19 | 83% | 91% |
| 30 residues | 29 | 75% | 86% |
| 44 residues | 43 | 65% | 81% |
That is theoretical full-length yield before any purification, and it explains several things at once. Long peptides cost more because more of the crude material is wrong. A tripeptide is close to trivial to make well; a forty-residue chain is not. And the difference between a coupling efficiency of 99% and 99.5% — which sounds like nothing — is the difference between two thirds and four fifths of the product being correct.
The longest chain in our range is Tesamorelin at 44 residues; the shortest are tripeptides at three. They are not remotely the same manufacturing problem.
Cleavage and purification
When the sequence is complete the chain is cut from the resin and the side-chain protection is removed, usually in the same step. What comes off is crude peptide: the target sequence plus the deletions, plus assorted side products.
Purification is then normally by preparative HPLC — the same separation principle used later to measure purity, run at scale to collect the target rather than merely to look at it. The purified material is then freeze-dried to a powder.
What this means when you read a certificate
A purity figure is the outcome of that whole process, and the impurities it accounts for are mostly near-relatives of the target — chains missing a residue, or carrying a leftover protecting group. They are not random contamination.
It also explains why the salt is there. Cleavage and purification both involve acids, and the peptide comes out as a salt of one of them — which is real mass in the vial that is not peptide, and invisible to the purity measurement.
The twenty amino acids · How to read a sequence · What is a peptide?
