TB-500 — 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 required
- Mobile phase and gradient
- A: 0.1 % trifluoroacetic acid in water; B: acetonitrile. Gradient 2–25 % B over 20 min — an extremely polar peptide requiring a near-aqueous starting condition
- Detection
- UV 214 nm only. No aromatic residue is present; 280 nm detection is impossible and a 280 nm purity figure is erroneous
- Retention
- Very early. Retention is so poor on conventional methods that void-region coelution is the principal source of overstated purity for this compound
§6.2Identity by mass spectrometry
[M+H]⁺ at m/z 861.5. Average mass 860.0 ± 1 Da. The distinction from full-length thymosin beta-4 at 4963 Da is unmistakable at any resolution, so substitution between the two is a labelling failure and not an analytical limitation.[3]
§6.3Related substances and degradation
Table 7. Related substances recorded for TB-500, with the process or storage route that generates each and its analytical signature.
| Related substance | Origin | Analytical signature |
|---|---|---|
| Full-length thymosin beta-4 | Substitution or mislabelling | +4103 Da — trivially distinguishable, yet the Institute has recorded this substitution |
| Non-acetylated LKKTETQ | Failed acetylation | −42 Da; the acetyl group is the only stabilising modification present and its absence is material |
| Des-Leu1 truncation | Incomplete coupling | −113 Da |
| Pyroglutamate at the C-terminal Gln | Cyclisation on storage | −17 Da |
| Counter-ion | Purification | Two lysines; counter-ion content is proportionally very large on an 860 Da peptide and may exceed 15 % by mass in poorly desalted material |
Degradation routes
- C-terminal glutamine cyclisation to pyroglutamate
- Deamidation of Gln7
- No oxidation-sensitive residues
- Aminopeptidase susceptibility in any biological matrix
§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; 2 mg is then 1.0 mL, that is 100 units on a U-100 syringe — the full barrel of a standard insulin syringe.
- Storage, lyophilised
- −20 °C, desiccated
- Storage, reconstituted
- 2–8 °C; no supported in-use claim
- In-use period
- No supported claim
The very large counter-ion fraction typical of small polybasic peptides means the peptide-content figure matters more here than the purity figure. A vial labelled 5 mg may contain appreciably less than 5 mg of peptide, entirely legitimately, and the certificate is where that is disclosed or is 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.