Semaglutide — 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, 100 Å pore
- Mobile phase and gradient
- A: 0.1 % trifluoroacetic acid in water; B: 0.1 % trifluoroacetic acid in acetonitrile. Gradient 30–60 % B over 25 min at 1.0 mL/min, 40 °C
- Detection
- UV 214 nm (amide backbone) with confirmatory 280 nm (Trp/Tyr)
- Retention
- Main peak elutes late relative to unmodified GLP-1 analogues because of the C18 diacid; typical retention 14–18 min under the stated conditions
§6.2Identity by mass spectrometry
ESI-MS in positive mode gives a charge envelope dominated by [M+3H]³⁺ at m/z ≈ 1372.2 and [M+4H]⁴⁺ at m/z ≈ 1029.4. Deconvolution should return an average mass within 2 Da of 4113.6. A monoisotopic determination requires resolution above 30,000 at m/z 1000.[3]
§6.3Related substances and degradation
Table 7. Related substances recorded for Semaglutide, with the process or storage route that generates each and its analytical signature.
| Related substance | Origin | Analytical signature |
|---|---|---|
| Des-amino truncation at the N-terminus | Deletion of His7 | −137 Da; elutes earlier |
| Aib8 → Ala8 sequence variant | Incorrect building block during synthesis | −14 Da; near-coeluting, requires MS to resolve |
| Free acid (non-acylated) backbone | Incomplete side-chain conjugation | −700 Da approximately; elutes substantially earlier and is the most diagnostic impurity of poor synthesis |
| Deamidated Asn/Gln forms | Storage or process-related | +1 Da; resolvable on a shallow gradient |
| Trifluoroacetate adduct | Residual counter-ion from purification | Not a peptide impurity; quantify separately by ion chromatography or ¹⁹F NMR |
Degradation routes
- Oxidation of the methionine-free sequence is not a principal route; tryptophan oxidation at Trp31 occurs under peroxide or light stress (+16 Da)
- Aspartate isomerisation to isoaspartate on prolonged aqueous storage
- Fibrillation and higher-order aggregation on agitation or at pH near the isoelectric point
- Hydrolysis of the C18 diacid linker under strongly acidic or alkaline conditions
§7Presentation, reconstitution and storage
§7.1Presentation and reconstitution
- Presentation
- Aqueous solution for injection in multi-dose pen or cartridge (approved products), or lyophilised powder in a sealed vial (research-supply material)
- Reconstitution
- For lyophilised material, add bacteriostatic or sterile water down the vial wall and swirl; do not shake. A 5 mg vial reconstituted with 2.0 mL gives 2.5 mg/mL, at which 0.25 mg is 0.10 mL, that is 10 units on a U-100 syringe.
- Storage, lyophilised
- 2–8 °C protected from light for the labelled shelf life; −20 °C for long-term storage of research material. Allow to reach room temperature before opening to avoid moisture ingress.
- Storage, reconstituted
- 2–8 °C; do not freeze after reconstitution
- In-use period
- Approved pens carry an in-use period of up to 56 days at 2–8 °C or below 30 °C depending on presentation. The Institute does not extend in-use claims to reconstituted research material, for which no stability-indicating data are published.
Semaglutide adsorbs to glass and to some plastics at low concentration; polysorbate 80 in approved formulations mitigates this. Reconstituted research material prepared without a surfactant may lose measurable content to container surfaces at concentrations below approximately 0.2 mg/mL.
§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.