SS-31 (elamipretide) — analytical characterisation
Chromatographic conditions, identity, related substances, presentation, reconstitution and in-use stability.
§6Analytical characterisation
§6.1Chromatographic conditions
- Column
- C18, 4.6 × 150 mm, 3.5 µm; a chiral or shallow-gradient method is required to confirm the D-arginine configuration
- Mobile phase and gradient
- A: 0.1 % trifluoroacetic acid in water; B: acetonitrile. Gradient 10–40 % B over 20 min
- Detection
- UV 214 nm; 275 nm (Dmt and Phe) — the 2,6-dimethyltyrosine chromophore is shifted relative to tyrosine, which is itself a useful identity indicator
- Retention
- Intermediate for a tetrapeptide, reflecting substantial aromatic content
§6.2Identity by mass spectrometry
[M+H]⁺ at m/z 640.4; [M+2H]²⁺ at m/z 320.7. Average mass 639.8 ± 0.5 Da.[3]
§6.3Related substances and degradation
Table 7. Related substances recorded for SS-31 (elamipretide), with the process or storage route that generates each and its analytical signature.
| Related substance | Origin | Analytical signature |
|---|---|---|
| L-Arg at position 1 | Incorrect building block | Isobaric — undetectable by mass and requiring a chiral method. For a compound whose entire mechanism depends on a specific aromatic-cationic geometry, configuration is not a fine detail |
| Tyrosine in place of 2,6-dimethyltyrosine | Wrong building block, or a cheaper substitution | −28 Da; readily detected and the single most important substitution to exclude, since Dmt is expensive and Tyr is not |
| C-terminal free acid | Incomplete amidation | +0.98 Da |
| Des-Arg1 | Incomplete coupling | −156 Da |
| Trifluoroacetate counter-ion | Purification | Arg and Lys on a 640 Da peptide give a proportionally very large counter-ion fraction |
Degradation routes
- Hydrolysis of the C-terminal amide
- Tyrosine-ring oxidation and nitration
- Diketopiperazine formation under thermal stress
- No methionine or cysteine, so the compound is comparatively oxidatively stable
§7Presentation, reconstitution and storage
§7.1Presentation and reconstitution
- Presentation
- Solution for injection (investigational); lyophilised powder in research supply
- Reconstitution
- A 10 mg vial with 2.0 mL gives 5 mg/mL; a 40 mg clinical-scale dose would require 8 mL and is not a research-syringe quantity, which the Institute notes as an example of a compound whose clinical dose does not map onto research-supply presentations.
- Storage, lyophilised
- −20 °C, desiccated
- Storage, reconstituted
- 2–8 °C
- In-use period
- No supported claim
The substitution of tyrosine for 2,6-dimethyltyrosine would reduce the cost of synthesis substantially and would produce a compound with a 28 Da mass deficit and materially different pharmacology. It is exactly the kind of substitution that a mass-spectrometric identity check catches and a purity-only certificate does not.
§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.