Quality and COAs

Why a lyophilised peptide is not dry: residual water and Karl Fischer

Freeze-drying removes the water a peptide was purified in; it does not produce an anhydrous solid. Residual water is weighed as peptide, it plasticises the powder enough to move its glass transition, and it is the only component of the vial that changes after the certificate was signed.

Written for Analytical chemists, lab operators, and quality reviewers · Updated August 5, 2026

Key points

A characterised synthetic peptide reference material measured 50.2 mg of water per gram
Loss on drying counts every volatile; Karl Fischer counts only water
Free cysteine thiols consume iodine and inflate the reported water content

Research-use boundary

This article is educational and limited to non-clinical research context, documentation practices, and terminology. It does not provide preparation, administration, dosing, treatment, diagnostic, cosmetic, or consumer-use guidance, and nothing here indicates that any material is suitable for human or animal use.

A freeze-dried peptide is roughly five percent water

Lyophilisation removes the water a peptide was purified in. It does not produce an anhydrous solid. When a metrology group characterised a synthetic glucagon reference material by mass balance, Karl Fischer titration returned 50.2 milligrams of water per gram of powder — just over five percent by weight — alongside 103.03 milligrams per gram of trifluoroacetate. The chromatographic figure for the same material was a purity above 98 percent. Both statements are accurate. Together they describe a powder in which more than fifteen percent of the weighed mass was never peptide, and about a third of that share was water.

Water is the one component that does not stay put

Counter-ion content is fixed when the material is purified. Residual solvent is fixed when it is dried. Water is not fixed at all. A lyophilised cake is a high-surface-area solid produced by subliming ice out of a frozen matrix, and that structure readily takes water back out of whatever atmosphere it meets. Bachem's technical guidance — supplier documentation rather than peer-reviewed literature — notes that hydrophilic sequences and peptides rich in basic residues can absorb considerable moisture. The consequence for a certificate is specific: every other number on the page stays true for the life of the lot, and the water figure is a timestamp.

Water does more than take up room

In a lyophilised humanised monoclonal antibody formulation studied across one to eight percent residual moisture, the glass transition temperature fell from about 80 degrees Celsius at one percent to about 25 degrees Celsius at eight percent. Below the glass transition a solid is a rigid glass in which molecular motion, and therefore most degradation, is slow; above it the matrix is rubbery and mobile. Water is a plasticiser, so a powder at the wet end of that range is not merely damp — it can be sitting at its own glass transition at bench temperature. The same study found chemical stability decreased as moisture rose in both the glassy and the rubbery state, with faster aspartate isomerisation in the high-moisture samples. That was a formulated antibody with its own excipients rather than a peptide salt, so the transferable part is the mechanism, not the numbers.

Drier is not automatically better

The intuition that a lyophilisation cycle should chase zero water does not survive contact with the data. Work on freeze-dried methionyl human growth hormone and tissue plasminogen activator used the Brunauer-Emmett-Teller method to estimate the water monolayer on each protein — the quantity that shields its polar groups — and found that samples dried below that monolayer showed opalescence when redissolved, a sign of physical instability. Samples at monolayer or multilayer moisture instead showed greater loss of biological stability under temperature stress. The authors concluded that an optimum residual moisture balances the two, rather than minimising water. A very low water figure on a certificate is a result to understand, not automatically a better one.

Loss on drying is a different measurement

USP General Chapter 731 determines the amount of volatile matter of ANY kind driven off under the specified conditions. That includes water, residual synthesis and purification solvent, and anything else that leaves at that temperature. USP General Chapter 921 covers water determination, and its Karl Fischer methods are a chemical reaction with water specifically. The chapters are explicit about the relationship: loss on drying is appropriate where water appears to be the only volatile constituent. On a peptide still carrying acetonitrile or methanol from purification, loss on drying reads higher than Karl Fischer, and the gap between the two cannot be attributed to water. Reporting both is informative; substituting one for the other is not.

What Karl Fischer actually measures

The underlying reaction is the oxidation of sulfur dioxide by iodine, which proceeds only in the presence of water and consumes one mole of iodine per mole of water. The volumetric form adds iodine as a standardised titrant. The coulometric form generates iodine electrochemically at the anode and measures the electrical charge required, which makes Faraday's law the calibration rather than a standardisation step: two moles of electrons per mole of iodine, one mole of iodine per mole of water, 96,485 coulombs per mole of electrons and 18.02 grams per mole of water give 10.71 coulombs per milligram of water. A published NIH protocol puts the coulometric determination range at 10 micrograms to 200 milligrams of water, which is why coulometric is the sensible variant for a milligram-scale sample holding a few percent water.

What makes a Karl Fischer result wrong on a peptide specifically

Free cysteine thiols reduce iodine directly. The titrant they consume is counted as though it had met water, so the reported water content reads high. Peer-reviewed work describes thiol-containing drug substances as not amenable to direct Karl Fischer titration and titrates the thiol first with a separate reagent, then the water.
The reaction wants a medium around pH 5 to 8. A peptide trifluoroacetate salt is acidic; pushed below that window the titration slows and the endpoint drifts. Supplier technical documentation recommends buffering with imidazole.
Sequences rich in basic residues push the medium the other way, where iodine disproportionation consumes titrant that never saw water. The same documentation recommends salicylic or benzoic acid for that case.
Atmospheric water enters during weighing and transfer. On a hygroscopic cake the specimen gains water while it is being prepared, and a rising blank drift is the signal that it is happening.
A dense cake may not release all its water into the titration solvent. Oven or gas extraction carries water into the cell rather than relying on the solid dissolving completely.

What it does to a concentration calculated by weight

Any calculation that converts a weighed mass into a molar concentration treats the entire mass as peptide. Counter-ion contributes the larger and more predictable share of the error. Water contributes a smaller share that is less predictable, because it varies not only between lots but between the day a vial was filled and the day it was opened. Both errors are systematic rather than random: they do not average out across replicates, they shift every measurement made from that stock in the same direction. The remedy is not a better balance. It is knowing the water content and the net peptide content of the specific material being weighed.

How to read it on a certificate

Whether water content is reported at all — an absent test is information, not a pass.
Which method produced it: Karl Fischer, and whether volumetric or coulometric, or loss on drying, which is not the same test.
The test date relative to when the vial was filled and sealed, since this is the one figure that moves afterwards.
Whether a stated net peptide content already accounts for water, or whether water and counter-ion are listed as separate components to be subtracted.
For sequences containing free cysteine, whether the method addressed thiol interference.

The short version

Lyophilised means the free water was sublimed away, not that the solid is dry. A characterised synthetic peptide carried about five percent water by mass. That water is weighed as though it were peptide, it plasticises the solid enough to move the glass transition, and it is the only thing in the vial that changes after the certificate was signed. It is measured by a reaction specific to water rather than by heating and weighing — and the difference between those two methods is the residual solvent nobody asked about.

Common questions

How much water is in a lyophilised peptide?

Freeze-drying removes free water but does not produce an anhydrous solid. In a published mass-balance characterisation of a synthetic glucagon reference material, Karl Fischer titration measured 50.2 mg of water per gram of powder — just over 5 percent by weight — alongside 103.03 mg/g of trifluoroacetate counter-ion. The exact figure varies with sequence and with how the material has been stored and handled, because a lyophilised cake re-adsorbs moisture from the air it is exposed to.

What is Karl Fischer titration?

Karl Fischer titration measures water through a chemical reaction in which iodine oxidises sulfur dioxide only in the presence of water, consuming one mole of iodine per mole of water. USP General Chapter 921 describes it as Method I, in a volumetric form that adds iodine as a standardised titrant and a coulometric form that generates iodine electrochemically and counts the charge instead. In the coulometric form the calibration is Faraday’s law rather than a standardisation step: 10.71 coulombs of charge corresponds to one milligram of water.

Is loss on drying the same as water content?

No. Loss on drying determines the amount of volatile matter of any kind driven off under the specified conditions, which includes residual solvent and anything else that leaves at that temperature, not water alone. Karl Fischer titration is a chemical reaction specific to water. For a material that still carries residual synthesis or purification solvent, loss on drying returns the higher figure, and the difference between the two methods cannot be attributed to water.

Does residual moisture affect the stability of a freeze-dried solid?

Yes, and not only in one direction. In a lyophilised monoclonal antibody formulation studied across 1 to 8 percent residual moisture, the glass transition temperature fell from about 80 degrees Celsius at 1 percent to about 25 degrees Celsius at 8 percent, and chemical stability decreased as moisture rose. But drying below a protein’s calculated water monolayer has been reported to cause physical instability, and published work on freeze-dried methionyl human growth hormone and tissue plasminogen activator concluded that an optimum residual moisture balances physical against biological stability rather than minimising water.

Why can a cysteine-containing peptide give a falsely high Karl Fischer result?

Free thiol groups reduce iodine directly. Because Karl Fischer quantifies water by the iodine consumed, titrant consumed by a thiol is counted as though it had reacted with water, so the reported water content reads high. Peer-reviewed work on thiol-containing drug substances describes them as not amenable to direct Karl Fischer titration and uses a two-step approach that titrates the thiol first with a separate reagent and then the water.

Sources

Purity determination of synthetic glucagon using a mass balance approach Analytical and Bioanalytical Chemistry (PMC7064584) Primary source for the measured water content (50.2 mg/g by Karl Fischer) and trifluoroacetate content (103.03 mg/g) of a characterised synthetic peptide.
Effect of moisture on the stability of a lyophilized humanized monoclonal antibody formulation Pharmaceutical Research (PubMed 11683251) Primary source for the glass transition temperature falling from about 80 to about 25 degrees Celsius across 1 to 8 percent residual moisture, and for chemical stability decreasing as moisture rises.
Determining the optimum residual moisture in lyophilized protein pharmaceuticals Developments in Biological Standardization (PubMed 1592175) Primary source for the water-monolayer concept and the finding that drying below the monolayer causes physical instability.
Water determination in drugs containing thiols International Journal of Pharmaceutics (PubMed 10547459) Peer-reviewed source for thiol-containing substances not being amenable to direct Karl Fischer titration, and for the two-step titration that addresses it.
USP General Chapter 921, Water Determination United States Pharmacopeia Compendial source defining Method I (titrimetric, including coulometric), Method II and Method III.
USP General Chapter 731, Loss on Drying United States Pharmacopeia Compendial source for loss on drying measuring volatile matter of any kind, and for directing the reader to Water Determination where water is the only volatile constituent.
Determination of Water Content using the Karl Fischer Coulometric Method NCI Nanotechnology Characterization Laboratory (NCBI Bookshelf NBK619777) Published NIH protocol giving the coulometric determination range of 10 micrograms to 200 milligrams of water.
Quality control of amino acids and peptides Bachem (supplier technical documentation) Supplier-tier source for water being determined by Karl Fischer and for hydrophilic and basic peptides absorbing considerable moisture.
Side reactions in Karl Fischer titration Metrohm (supplier technical documentation) Supplier-tier source for iodine reduction by thiols giving high results, and for buffering acidic and basic samples into the working pH window.