Water content and stability of peptide powders

Why does a dry powder contain water?

A freeze-dried, or lyophilised, cake looks dry but can take up water from the air whenever it is exposed. The EMA guideline on synthetic peptides puts it plainly: "Peptides are often very hygroscopic powders", and it asks manufacturers to state hygroscopicity, for example with moisture sorption isotherms (EMA, Guideline on the Development and Manufacture of Synthetic Peptides).

Hygroscopic means the material pulls water vapour out of the surrounding air. A closed vial with an intact stopper limits that; an opened vial or a damaged stopper exposes the cake to room air. Lesson 5 showed that powder holds counterions and water as well as peptide. Water is the part most likely to change after the vial leaves the manufacturer.

How does Karl Fischer titration measure water?

Karl Fischer titration, usually shortened to KF, relies on a chemical reaction in which water reacts with iodine and sulfur dioxide. In the coulometric version, iodine is generated electrically and consumed by the water in the sample, and the instrument counts how much was needed until the water is used up (Wang et al., Scientific Reports, 2020). The result is reported as a percentage of the sample mass, or in mg per g.

KF is specific to water, which is its main advantage over a simple loss on drying test. Loss on drying measures any weight lost on heating, so it cannot tell water from a volatile solvent such as those covered in lesson 9. The USP reference standards team measured water in peptide reference materials "by Karl Fischer titration as described in USP general chapter <921> Water Determination" (McCarthy et al., Pharmaceutical Research, 2023). The Karl Fischer method page sets out the volumetric and coulometric versions in more detail.

The measurement has to fight the problem it measures. In the glucagon study, analysts weighed samples in a glovebox held within about 1% humidity with high-purity nitrogen, to stop the powder absorbing water during handling. Three replicates gave 51.0, 50.2 and 49.7 mg per g, an average of 50.2 mg per g, about 5% of the powder by mass. That is one reference material, not a typical value, but it shows the scale water can reach.

Why does water change the number of milligrams?

Every milligram of water is a milligram that is not peptide. If a label was set by weighing powder without allowing for water and counterions, the peptide mass will be lower than the weighed mass (lesson 4).

The USP team expressed this with a simple correction: water-corrected purity equals purity multiplied by (100 minus percent water), divided by 100 (McCarthy et al., 2023). An illustrative example, with numbers chosen only to show the arithmetic:

Counterions reduce the peptide share further. The content calculator runs this arithmetic for figures printed on a certificate. A certificate showing high purity but no water figure has not told you how much of the powder is peptide; a direct amount result against a reference standard sidesteps the question by measuring the peptide itself.

Water also matters for the reference standard. The EMA guideline says that precautions against moisture uptake by the reference standard "during storage and during analysis" should be taken, and that moisture content may need to be confirmed before use. A reference standard that has quietly gained water will make every sample measured against it look richer than it is. Lesson 13 returns to this.

What does stability mean in ICH Q1A terms?

ICH Q1A(R2) is the core guideline on stability. Its stated purpose is to provide evidence on "how the quality of a drug substance or drug product varies with time under the influence of a variety of environmental factors such as temperature, humidity, and light", and to set a re-test period or shelf life and storage conditions (ICH Q1A(R2)).

For peptides specifically, the EMA guideline notes that storage is generally refrigerated (5 degrees C plus or minus 3) or frozen (minus 20 degrees C plus or minus 5), lists degradation pathways such as "oxidation of Cys and Met residues, deamidation, hydrolysis", and says that "for hygroscopic powders, it is expected that water content should be part of the stability protocols."

Why does a hot, humid climate like the Philippines matter here?

Q1A says it "addresses climatic zones I and II" and was written for the EC, Japan and the United States. The World Health Organization's stability guideline describes how several countries in climatic zone IV, the hot and humid zone, moved to "up to 30 °C/75% RH as the long-term storage conditions for hot and humid regions", and notes that zone IVa is 30 degrees C at 65% RH while zone IVb is 30 degrees C at 75% RH (WHO TRS 1010, Annex 10). The same guideline notes that the less humid IVa condition may be accepted instead when humidity is not an issue, for example for storage in glass containers.

Two points follow for vials in a tropical climate. Because a sealed glass vial keeps out humid air, the integrity of the stopper and crimp matters more than the room. And time a vial spends warm, in a parcel, a vehicle or on a counter, is outside anything a certificate can describe.

How should I read water and stability lines on a certificate?

• Water (KF): x%. A specific water figure. Note the date: it describes the powder on that day, not after months on a shelf.
• Loss on drying: x%. Weight lost on heating. It includes water and any volatile solvent, so it is not the same as a KF result.
• No water line at all. Common, and the reason purity and amount should not be confused. Look for a direct amount result instead.
• A stated shelf life, such as "stable for 24 months". Ask for the study behind it: which conditions, how many batches, which tests.
• A re-test or expiry date. Under Q1A this should rest on long-term data at the stated storage condition.

What does this mean for comparing results over time?

Because water uptake and degradation depend on time and conditions, two results for the same batch analysed months apart can legitimately differ without either lab being wrong. That is why every result should carry an analysis date. The semaglutide and tirzepatide pages cover compound-specific failure modes, and lesson 11 explains why one vial cannot speak for a whole batch even on the day it is analysed.

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