Lixisenatide — 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 high-pH-tolerant phase assists resolution of the basic polylysine tail
- Mobile phase and gradient
- A: 0.1 % trifluoroacetic acid in water; B: acetonitrile. Gradient 18–40 % B over 30 min
- Detection
- UV 214 nm
- Retention
- Earlier than exenatide; the polylysine tail reduces hydrophobic retention
§6.2Identity by mass spectrometry
[M+4H]⁴⁺ at m/z ≈ 1215.6; deconvoluted average mass 4858.5 ± 2.5 Da. The six lysines raise the average charge state and shift the envelope to higher charge than exenatide.[3]
§6.3Related substances and degradation
Table 7. Related substances recorded for Lixisenatide, with the process or storage route that generates each and its analytical signature.
| Related substance | Origin | Analytical signature |
|---|---|---|
| Des-Lys truncations | Incomplete coupling in the polylysine tail | −128 Da per residue; produces a characteristic ladder |
| Met14 sulfoxide | Oxidation | +16 Da |
| C-terminal free acid | Incomplete amidation | +0.98 Da |
| Trifluoroacetate | Counter-ion | Elevated in basic peptides; quantify separately — the polylysine tail binds counter-ion stoichiometrically and TFA content of 8–12 % by mass is not unusual in poorly desalted material |
Degradation routes
- Methionine oxidation
- Deamidation
- Truncation of the polylysine tail on prolonged storage in solution
§7Presentation, reconstitution and storage
§7.1Presentation and reconstitution
- Presentation
- Aqueous solution in pen; fixed combination with insulin glargine
- Reconstitution
- Rarely required; research supply is uncommon
- Storage, lyophilised
- 2–8 °C
- Storage, reconstituted
- 2–8 °C
- In-use period
- Approved pen: 14 days below 30 °C after first use
The high counter-ion content typical of basic polylysine-containing peptides is the reason the Institute treats counter-ion quantification as a critical, not optional, certificate field for this structural class.
§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.