DSIP — 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; aqueous-compatible phase preferred
- Mobile phase and gradient
- A: 0.1 % trifluoroacetic acid in water; B: acetonitrile. Gradient 3–30 % B over 20 min
- Detection
- UV 214 nm; 280 nm strong relative to peptide size (single Trp1) — the 280/214 ratio is a useful identity indicator here
- Retention
- Early; the acidic sequence and small size give poor retention
§6.2Identity by mass spectrometry
[M+H]⁺ at m/z 849.3; [M−H]⁻ at m/z 847.3 in negative mode, which suits this acidic sequence well.[3]
§6.3Related substances and degradation
Table 7. Related substances recorded for DSIP, with the process or storage route that generates each and its analytical signature.
| Related substance | Origin | Analytical signature |
|---|---|---|
| Trp1 oxidation products | Oxidation and light exposure | +16 and +32 Da; an N-terminal tryptophan is both oxidation- and photolysis-labile |
| Aspartimide at Asp5 | Base-mediated side reaction | −18 Da |
| Des-Trp1 | Aminopeptidase cleavage or incomplete coupling | −186 Da; also removes the only chromophore, so a preparation dominated by this impurity would show anomalously low 280 nm absorbance |
| Pyroglutamate at the C-terminal Glu | Cyclisation | −18 Da |
| Sodium or ammonium counter-ion | This is an acidic peptide, so the counter-ion is a cation rather than an anion — a point suppliers frequently get wrong when reporting "TFA content" for acidic sequences |
Degradation routes
- Tryptophan oxidation and photolysis
- Aspartimide formation at Asp5
- Peptide-bond hydrolysis
§7Presentation, reconstitution and storage
§7.1Presentation and reconstitution
- Presentation
- Lyophilised powder in vial (research supply only)
- Reconstitution
- A 5 mg vial with 2.5 mL gives 2 mg/mL; 100 µg is 0.05 mL, that is 5 units on a U-100 syringe.
- Storage, lyophilised
- −20 °C, desiccated, protected from light
- Storage, reconstituted
- 2–8 °C protected from light
- In-use period
- No supported claim
DSIP is an acidic peptide, which has a practical consequence the Institute notes because it is widely misunderstood: acidic peptides are supplied as cation salts, and a certificate reporting trifluoroacetate content for an acidic peptide with no basic residues indicates that the counter-ion determination was not actually performed.
§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.