Thymosin alpha-1 — 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
- Mobile phase and gradient
- A: 0.1 % trifluoroacetic acid in water; B: acetonitrile. Gradient 10–40 % B over 30 min
- Detection
- UV 214 nm only — the sequence contains no tryptophan, tyrosine or phenylalanine, so it has no usable 280 nm chromophore. This is the third compound in the series for which a reported 280 nm purity would be erroneous
- Retention
- Early to intermediate; a highly acidic sequence
§6.2Identity by mass spectrometry
[M+3H]³⁺ at m/z ≈ 1037.1; [M−2H]²⁻ at m/z ≈ 1553.2 in negative mode, which suits the acidic sequence. Average mass 3108.3 ± 2 Da.[3]
§6.3Related substances and degradation
Table 7. Related substances recorded for Thymosin alpha-1, with the process or storage route that generates each and its analytical signature.
| Related substance | Origin | Analytical signature |
|---|---|---|
| Non-acetylated N-terminus | Failed acetylation | −42 Da; N-terminal acetylation is part of the definition of the molecule and its absence is an identity failure rather than a purity finding |
| Deamidated Asn28 | Storage | +1 Da; the C-terminal asparagine is the principal deamidation site |
| Aspartimide at Asp2, Asp6 or Asp15 | Base-mediated side reaction | −18 Da; three aspartate residues make this a recurrent synthesis issue |
| Truncated chains | Incomplete coupling in a 28-mer | Multiple species |
| Cation counter-ion | Strongly acidic peptide | Sodium or ammonium; a trifluoroacetate determination is inapplicable |
Degradation routes
- Deamidation of Asn28
- Aspartimide formation at three sites
- No oxidation-sensitive residues, so the compound is oxidatively stable
- Aggregation is not a prominent route for this highly charged acidic sequence
§7Presentation, reconstitution and storage
§7.1Presentation and reconstitution
- Presentation
- Lyophilised powder for reconstitution (approved product supplied with diluent)
- Reconstitution
- The approved presentation is 1.6 mg reconstituted with 1.0 mL of the supplied diluent, giving 1.6 mg/mL; the full 1.6 mg dose is then 1.0 mL. Research vials are commonly 5 or 10 mg; a 10 mg vial with 5.0 mL gives 2 mg/mL and a 1.6 mg dose of 0.80 mL, that is 80 units on a U-100 syringe.
- Storage, lyophilised
- 2–8 °C for the approved product; −20 °C for research material
- Storage, reconstituted
- The approved label directs use immediately after reconstitution
- In-use period
- Immediate use per the approved label
Thymosin alpha-1 is the compound in this series for which the gap between a real marketing authorisation and the claims made in research supply is narrowest — but the authorised indications are hepatitis and vaccine adjuvancy, not general immune enhancement, and the Institute states that distinction.
§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.