Pramlintide — analytical characterisation
Chromatographic conditions, identity, related substances, presentation, reconstitution and in-use stability.
§6Analytical characterisation
§6.1Chromatographic conditions
- Column
- C18, 4.6 × 250 mm, 5 µm
- Mobile phase and gradient
- A: 0.1 % trifluoroacetic acid in water; B: acetonitrile. Gradient 15–40 % B over 30 min
- Detection
- UV 214 nm; 275 nm weak (single Tyr, single Phe pair)
- Retention
- Early relative to lipidated analogues
§6.2Identity by mass spectrometry
[M+3H]³⁺ at m/z ≈ 1317.5; deconvoluted average mass 3949.4 ± 2 Da. Reduction of the disulfide adds 2 Da and is diagnostic of a reduced or scrambled form.[3]
§6.3Related substances and degradation
Table 7. Related substances recorded for Pramlintide, with the process or storage route that generates each and its analytical signature.
| Related substance | Origin | Analytical signature |
|---|---|---|
| Reduced (free-thiol) form | Failed or reversed disulfide formation | +2 Da; biologically inactive and a critical finding |
| Disulfide-scrambled dimer | Intermolecular oxidation | Approximately double mass; detected by size-exclusion or non-reducing electrophoresis |
| Des-Lys1 metabolite or process impurity | Truncation | −128 Da; retains activity, complicating potency interpretation |
| Deamidated Asn/Gln forms | Storage | +1 Da — this sequence contains five asparagine and two glutamine residues and is unusually deamidation-prone |
Degradation routes
- Deamidation is the dominant aqueous route given the high asparagine content
- Disulfide scrambling and dimerisation
- Despite the proline substitutions, aggregation remains possible at low pH
§7Presentation, reconstitution and storage
§7.1Presentation and reconstitution
- Presentation
- Aqueous solution in pen; lyophilised powder in research supply
- Reconstitution
- A 5 mg vial with 5.0 mL gives 1 mg/mL; a 60 µg dose is then 0.06 mL, that is 6 units on a U-100 syringe.
- Storage, lyophilised
- 2–8 °C
- Storage, reconstituted
- 2–8 °C, do not freeze
- In-use period
- Approved pen: 30 days at up to 30 °C after first use
The disulfide bond makes this one of the few peptides in the series for which a non-reducing versus reducing comparison is an essential identity check.
§7.2In-use stability
Applicable standards: CEI-MS-01 · CEI-MS-02 · CEI-MS-03 · CEI-MS-04 · CEI-MS-05 · CEI-MS-06. The full series is at methodological standards.
Working calculators: reconstitution and insulin-unit conversion · purity against peptide content · certificate minimum-data checker.
References cited on this page
References are numbered in order of first citation in this document. Each superscript in the text links to its entry below.
- United States Pharmacopeial Convention. General Chapter ⟨1225⟩ Validation of Compendial Procedures. United States Pharmacopeia — National Formulary (USP–NF) 2024;USP 2024 Issue 1. identifier not held by the Institute
- International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. ICH Q2(R2) Validation of Analytical Procedures. ICH Harmonised Guideline 2023;Step 4 version, 1 November 2023. identifier not held by the Institute
- International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. ICH Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products. ICH Harmonised Tripartite Guideline 1999;Step 4 version. identifier not held by the Institute
- International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. ICH Q1A(R2) Stability Testing of New Drug Substances and Products. ICH Harmonised Tripartite Guideline 2003;Step 4 version. identifier not held by the Institute
- Manning MC, Chou DK, Murphy BM, Payne RW, Katayama DS. Stability of protein pharmaceuticals: an update. Pharmaceutical Research 2010;27(4):544–575. doi:10.1007/s11095-009-0045-6 · PMID 20143256
Identifiers are reproduced only where the Institute holds them. Where a digital object identifier or PubMed identifier is not shown, the Institute has recorded the journal and year and has not constructed an identifier.