Source of Reconstitution of lyophilised peptides

{{Infobox method | name = Reconstitution of lyophilised peptides | subtitle = Preparation | image = vial.svg | caption = Diluent is directed down the vial wall rather than onto the cake. | Reverses = [[Lyophilisation|Lyophilisation]] | Common diluents = [[Bacteriostatic water]], [[Sterile water for injection]] | Consequence = Solution stability is far shorter than powder stability }} {{hatnote|For the calculator, see [[Reconstitution calculator]]. For the drying process this reverses, see [[Lyophilisation]].}} '''Reconstitution''' is the dissolution of a [[Lyophilisation|lyophilised]] peptide cake in a liquid diluent. The dried solid is the stable form: once in solution the peptide is exposed to hydrolysis, [[Deamidation|deamidation]], oxidation and [[Peptide aggregation|aggregation]], all of which proceed far faster in the liquid phase.{{r|manning2010}} 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 [[Peptide content|content]], water and fill mass are known — a vial nominally containing 5 mg may contain appreciably less peptide. See [[Underfilling]].{{r|usp1503}} 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.{{r|usp797}} == Mechanics == 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.{{r|manning2010}} 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]].{{r|usp1503}} == Concentration arithmetic == 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.{{r|usp1503}} | Nominal fill | Diluent | Nominal concentration | |---|---|---| | 5 mg | 1.0 mL | 5 mg/mL | | 5 mg | 2.0 mL | 2.5 mg/mL | | 10 mg | 2.0 mL | 5 mg/mL | | 2 mg | 1.0 mL | 2 mg/mL | 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, [[Peptide content|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 [[Reconstitution calculator|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 [[Insulin syringe|syringe]] graduations, which are marked in insulin units rather than in millilitres. See [[Insulin syringe unit conversion]].{{r|usp1503}} == Storage after reconstitution == 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.{{r|ich_q1a}} 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.{{r|manning2010}} Where a preservative is absent, the limiting factor is microbiological rather than chemical, and it applies from the first entry of the container. Research-chemical powders are not sterile products — see [[Sterility testing]] — so a reconstituted solution is not a sterile solution regardless of diluent. Nothing here is medical advice, and research-use compounds are not approved for human administration; see [[Beyond-use date]] for how dating is assigned in settings where it is formally required.{{r|usp797}} == References == {{reflist}} <ref name="manning2010">Manning MC, Chou DK, Murphy BM, Payne RW, Katayama DS. "Stability of protein pharmaceuticals: an update." ''Pharmaceutical Research'' 27(4):544–575 (2010). PMID 20143256.</ref> <ref name="usp1503">United States Pharmacopeia, General Chapter <1503>, ''Quality Attributes of Synthetic Peptide Drug Substances''.</ref> <ref name="usp797">United States Pharmacopeia, General Chapter <797>, ''Pharmaceutical Compounding — Sterile Preparations''.</ref> <ref name="ich_q1a">International Council for Harmonisation, ''Q1A(R2): Stability Testing of New Drug Substances and Products'' (2003).</ref> == See also == * [[Lyophilisation]] * [[Bacteriostatic water]] * [[Sterile water for injection]] * [[Beyond-use date]] * [[Peptide aggregation]] * [[Reconstitution calculator]] {{DEFAULTSORT:Reconstitution of lyophilised peptides}} [[Category:Preparation and handling]] [[Category:Clinical practice]] [[Category:Cold chain and stability]]