Reconstitution of lyophilised peptides (revision 24)

Reconstitution is the dissolution of a lyophilised peptide cake in a liquid diluent. The dried solid is the stable form: once in solution the peptide is exposed to hydrolysis, deamidation, oxidation and aggregation, all of which proceed far faster in the liquid phase.^[1]

Two quantities determine the resulting concentration: the mass of peptide actually present in the vial, and the volume of diluent added. The first is not the number printed on the label unless content, water and fill mass are known — a vial nominally containing 5 mg may contain appreciably less peptide. See Underfilling.^[2]

The choice of diluent determines how long the resulting solution can be regarded as usable. Bacteriostatic water contains a preservative and is used where a container will be entered more than once; Sterile water for injection contains none. Neither confers sterility on a non-sterile powder.^[3]

Diluent is directed against the vial wall rather than onto the cake. A jet striking the cake dissolves it violently, generating the air–liquid interfaces and shear at which peptides unfold and aggregate; running the liquid down the wall lets the cake dissolve from beneath.^[1]

Dissolution is completed by gentle swirling or by leaving the vial to stand. Shaking is the practice most likely to produce visible haze, for the same interfacial reason. A cake that has not fully dissolved after several minutes should be given time rather than agitation.

Visual inspection after dissolution is informative. A clear, colourless solution free of visible particles is the expected result; haze, particles or fibrous material indicate that the material has changed, whatever the certificate said about the powder. See Peptide aggregation.^[2]

Concentration is peptide mass divided by diluent volume. The volume displaced by a few milligrams of solid is negligible at ordinary scales, so added volume can be treated as final volume without material error.^[2]

These are nominal figures computed from the label claim. The actual concentration depends on the mass of peptide in the vial, which is the product of fill mass, content and purity — a vial of 5 mg solid at 80% content holds 4 mg of peptide, so the first row is really 4 mg/mL. The calculator article carries a working tool; the arithmetic itself is trivial and the difficulty is entirely in knowing the numerator.

Volumes drawn from the resulting solution are governed by syringe graduations, which are marked in insulin units rather than in millilitres. See Insulin syringe unit conversion.^[2]

A reconstituted solution has a much shorter usable life than the powder it came from. Degradation routes that are negligible in the dry solid — deamidation, oxidation, aggregation, and microbial growth where no preservative is present — all operate.^[4]

Refrigeration slows chemical degradation substantially: rate constants for deamidation and oxidation fall several-fold between ambient and 2–8 °C. Freezing a reconstituted peptide solution is generally counterproductive, since ice formation concentrates solutes and creates interfaces, and repeated freeze–thaw is a well-documented cause of aggregation.^[1]

• Lyophilisation
• Bacteriostatic water
• Sterile water for injection
• Beyond-use date
• Peptide aggregation
• Reconstitution calculator