Reference
What the milligrams on a peptide vial mean
A vial marked 10mg does not contain 10mg of peptide. That is not a defect and not a trick — it is what the number has always referred to.
Almost every complaint about short-filled vials in this field is a misunderstanding of what the label number is a measurement of. There are three different weights involved and they are never the same as one another.
Three numbers, not one
| Number | What it measures |
|---|---|
| Fill weight | Everything that went into the vial: peptide, counterion, residual water, residual solvent, any excipient. |
| Nominal content | What the label says — 10mg, 5mg. This is what was specified, not what was weighed. |
| Net peptide content | The weight of actual peptide backbone in there, as a percentage. Typically somewhere between 70% and 90%. |
Where the missing weight goes
The counterion. A peptide with basic residues comes off purification as a salt, usually a trifluoroacetate, occasionally an acetate. Those counterions are real matter with real mass, and on a short peptide with several basic residues they can be a fifth of the powder. This is covered at length on why a peptide is a salt, and it is the largest single component of the gap.
Residual moisture. Freeze-drying removes most of the water, not all of it. A well-run process leaves a few percent, and a peptide cake left in humid air will take more back on. See what lyophilisation actually does.
Residual solvent. Acetonitrile and similar are used in purification and are mostly but not entirely driven off.
Excipients, where present. Some preparations include a bulking agent such as mannitol to give the cake structure. Where they are used they are a declared part of the fill and a large part of the weight.
Purity and net peptide content are different numbers
This is the confusion worth clearing up, because the two are routinely printed on the same document and read as if they were one figure.
- Purity — of the peptide material present, how much of it is the right peptide. A 99% purity figure says 1% of the peptide in there is some other peptide. It says nothing at all about counterion or water, because the method it comes from cannot see them. See what an HPLC purity figure measures.
- Net peptide content — of the total powder in the vial, how much is peptide of any kind. Determined by amino acid analysis or by nitrogen determination, not by HPLC.
A vial can honestly be 99% pure and 78% peptide by weight at the same time. Both numbers are correct and they are answering different questions.
Why two vials both marked 10mg can differ
Two suppliers filling to the same nominal 10mg can deliver measurably different quantities of peptide, without either of them doing anything wrong, if:
- one supplies a trifluoroacetate salt and the other an acetate;
- one's lyophilisation leaves 1% residual moisture and the other's leaves 5%;
- one fills to nominal peptide content and the other fills to gross weight.
That last one is the one to ask about. Filling to net peptide content means weighing out enough powder that the peptide in it comes to 10mg, which requires knowing the net peptide content first. Filling to gross weight means putting 10mg of powder in. They are different amounts of peptide and the label looks identical either way.
What a vial should look like
Ten milligrams of lyophilised peptide spread across the bottom of a 3ml vial is a very small amount of material — often a thin film, sometimes a smear, occasionally nothing you can see against a white background. A vial that looks empty at 10mg is normal and a vial with a visible cake at 10mg usually contains a bulking agent.
Why a peptide is a salt · Working out a molecular weight · Glossary
