PT-141 (bremelanotide) — 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 15–45 % B over 25 min
- Detection
- UV 214 nm; 280 nm strong (Trp) — a reliable secondary wavelength for this compound
- Retention
- Intermediate; the cyclic constraint reduces retention relative to the linear analogue, which is itself a useful distinguishing feature
§6.2Identity by mass spectrometry
[M+H]⁺ at m/z 1026.5; [M+2H]²⁺ at m/z 513.8. Average mass 1025.2 ± 1 Da.[3]
§6.3Related substances and degradation
Table 7. Related substances recorded for PT-141 (bremelanotide), with the process or storage route that generates each and its analytical signature.
| Related substance | Origin | Analytical signature |
|---|---|---|
| Linear (uncyclised) heptapeptide | Failed lactam formation | +18 Da; the linear form lacks the conformational constraint that produces the pharmacology and is a critical impurity. It is also chromatographically resolvable, so a laboratory that reports a single peak without addressing cyclisation has not confirmed the structure |
| Melanotan-II | Substitution or synthesis of the wrong analogue | −1 Da relative to bremelanotide, since melanotan-II is the C-terminally amidated form. A 1 Da difference on a 1025 Da molecule requires high-resolution mass spectrometry to resolve, and the two compounds have very different regulatory status. The Institute regards this as one of the most consequential near-isobaric pairs in the field |
| Trp6 oxidation | Oxidation | +16 Da |
| D-Phe to L-Phe at position 4 | Incorrect building block | Isobaric; requires chiral analysis. The D-configuration is essential to melanocortin receptor affinity |
| Trifluoroacetate counter-ion | Purification | Arg, His and Lys are present; content should be quantified |
Degradation routes
- Tryptophan oxidation
- Hydrolysis of the lactam bridge, regenerating the linear peptide
- Deamidation is not applicable to the free-acid C-terminus
- Aspartimide formation is prevented by the involvement of the Asp side chain in the lactam
§7Presentation, reconstitution and storage
§7.1Presentation and reconstitution
- Presentation
- Solution in single-dose autoinjector (approved product); lyophilised powder in research supply
- Reconstitution
- A 10 mg vial with 2.0 mL gives 5 mg/mL; the approved 1.75 mg dose is then 0.35 mL, that is 35 units on a U-100 syringe.
- Storage, lyophilised
- 2–8 °C for the approved product; −20 °C for research material
- Storage, reconstituted
- 2–8 °C protected from light
- In-use period
- The approved presentation is single-dose. No in-use claim is published for research material
The one-dalton difference between bremelanotide and melanotan-II is the single most instructive analytical fact in this pair of monographs. One compound is an approved medicine with a defined dosing limit; the other has never been approved anywhere and carries a melanoma concern. A low-resolution mass spectrum cannot tell them apart.
§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.