Thymosin beta-4 — analytical characterisation
Chromatographic conditions, identity, related substances, presentation, reconstitution and in-use stability.
§6Analytical characterisation
§6.1Chromatographic conditions
- Column
- C18 or C8, 4.6 × 250 mm, 5 µm, 300 Å wide pore for a 43-mer
- Mobile phase and gradient
- A: 0.1 % trifluoroacetic acid in water; B: acetonitrile. Gradient 10–40 % B over 40 min
- Detection
- UV 214 nm; 258 nm weak (single Phe at position 12; no Trp or Tyr) — so 280 nm detection is effectively unusable and this is a second peptide in the series for which a 280 nm purity claim is not credible
- Retention
- Intermediate for a hydrophilic 43-mer
§6.2Identity by mass spectrometry
[M+4H]⁴⁺ at m/z ≈ 1241.9; average mass 4963.4 ± 3 Da. Met6 oxidation adds 16 Da.[3]
§6.3Related substances and degradation
Table 7. Related substances recorded for Thymosin beta-4, with the process or storage route that generates each and its analytical signature.
| Related substance | Origin | Analytical signature |
|---|---|---|
| Met6 sulfoxide | Oxidation | +16 Da; the principal degradation impurity |
| Non-acetylated N-terminus | Failed acetylation | −42 Da |
| TB-500 heptapeptide | Substitution | −4103 Da; unmistakable |
| Deamidated Asn23 and Gln residues | Storage | +1 Da each; the sequence contains one asparagine and five glutamines |
| Truncated chains | Incomplete coupling in a 43-residue synthesis | Multiple species; crude purity for a 43-mer is substantially lower than for short peptides, so a high final purity implies extensive purification |
Degradation routes
- Methionine oxidation
- Deamidation at multiple glutamine sites
- Aggregation at concentration
- Aspartimide formation at the Asp-Lys-Pro-Asp N-terminal region
§7Presentation, reconstitution and storage
§7.1Presentation and reconstitution
- Presentation
- Lyophilised powder in vial
- Reconstitution
- A 10 mg vial with 2.0 mL gives 5 mg/mL; 2 mg is then 0.40 mL, that is 40 units on a U-100 syringe.
- Storage, lyophilised
- −20 °C, desiccated
- Storage, reconstituted
- 2–8 °C; no supported in-use claim
- In-use period
- No supported claim
Because the sequence has almost no aromatic content, this peptide is a good illustration of why UV 214 nm is the mandatory detection wavelength for peptide purity and 280 nm is a supplementary confirmation applicable only to aromatic-containing sequences.
§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.