1.0 Identification
| Common Name | TB-500; Thymosin Beta-4 Fragment (17–23) |
|---|---|
| IUPAC Name | N-acetyl-L-seryl-L-α-aspartyl-L-lysyl-L-prolyl-L-α-aspartyl-L-methionyl-L-alanyl-L-glutamic acid |
| CAS Number | 77591-33-4 |
| Molecular Formula | C41H68N10O16S |
| Molecular Weight | 4963.44 g/mol (average); monoisotopic mass 4960.42 Da |
| Sequence (three-letter) | Ac-Ser-Asp-Lys-Pro-Asp-Met-Ala-Glu |
| Sequence (one-letter) | Ac-SDKPDMAE |
| Parent Protein | Thymosin Beta-4 (Tβ4); full-length protein is a 43-amino-acid polypeptide encoded by the TMSB4X gene |
| Terminus | N-terminal acetyl group; C-terminus free acid |
Amino acid sequence in single-letter notation with N-terminal acetylation:
TB-500 corresponds to residues 17–23 of the full-length Thymosin Beta-4 protein, specifically the actin-binding domain. The discrete octapeptide is not isolated from biological tissue in significant quantities; all analytical-grade material is produced by chemical synthesis.
2.0 Source
Thymosin Beta-4 was first characterized by Low et al. (1981) as a thymic hormone fraction with actin-binding activity [1]. The actin-binding domain, corresponding to the Ac-SDKPDMAE octapeptide, was subsequently delineated by Huff et al. (2001) using deletion analysis and NMR spectroscopy [2]. The abbreviated peptide received the designation TB-500 in the research literature to distinguish it from the full-length 43-residue protein. Nexphoria TB-500 is produced exclusively by solid-phase chemical synthesis; no biological extraction is involved in manufacture.
3.0 Synthesis Notes
TB-500 is synthesized by Fmoc solid-phase peptide synthesis (Fmoc-SPPS) on Wang resin, which provides a C-terminal free acid upon cleavage. N-terminal acetylation is performed on-resin prior to global deprotection. Coupling employs HATU/DIPEA activation in NMP at a 3× molar excess per residue to ensure complete incorporation. Fmoc removal is performed with 20% piperidine in DMF.
| Synthesis Parameter | Value |
|---|---|
| Strategy | Fmoc-SPPS, stepwise C→N assembly |
| Resin | Wang resin, 0.4–0.6 mmol/g loading; yields C-terminal free acid |
| Coupling reagent | HATU / DIPEA, 3× molar excess per residue |
| Fmoc deprotection | 20% piperidine in DMF, 2 × 10 min |
| N-terminal acetylation | Acetic anhydride / DIPEA in DMF, 30 min, on-resin prior to global deprotection |
| Cleavage cocktail | TFA:TIPS:H₂O (95:2.5:2.5 v/v), 2–3 h at ambient temperature |
| Purification | Preparative RP-HPLC, C18 column (22×250 mm, 10 µm), 0.1% TFA in water/acetonitrile gradient |
| Purity specification | ≥98% by analytical HPLC (UV 220 nm, area percent) |
| Final form | Lyophilized powder; TFA counter-ion unless otherwise specified |
Following cleavage, the crude peptide is precipitated into cold diethyl ether, filtered, and redissolved in 0.1% aqueous TFA for preparative chromatography. Peak fractions are pooled, diluted, and lyophilized. Identity is confirmed by ESI-MS prior to lot release. Methionine-containing peptides (position 6) are monitored for sulfoxide formation; lots exceeding 0.5% Met(O) by-product are rejected [3].
4.0 Stability
Stability data are derived from internal real-time and accelerated studies in accordance with ICH Q1A(R2) guidelines. The methionine residue at position 6 is the primary degradation liability; oxidation to methionine sulfoxide is the dominant degradation pathway under aerobic aqueous conditions [3, 4].
| Condition | Duration | Specification |
|---|---|---|
| Lyophilized, −20°C, nitrogen atmosphere, dark | 24 months | ≥95% purity by HPLC; Met(O) <0.5% |
| Lyophilized, 4°C, dark | 12 months | ≥95% purity by HPLC |
| Reconstituted in bacteriostatic water, 2–8°C | 14 days | ≥90% purity; no visible turbidity; Met(O) <1% |
| Reconstituted, −20°C (single-use aliquots) | 30 days | ≥90% purity; limit to three freeze-thaw cycles |
| Reconstituted, 25°C, ambient atmosphere | 48 hours | Use within 48 h; protect from direct light; discard if turbid |
5.0 Storage & Handling
Storage conditions comply with USP <659> (Packaging and Storage Requirements). Vials are headspace-purged with dry nitrogen prior to capping to retard oxidative degradation of the methionine residue.
| Packaging | Type I borosilicate glass vial, bromobutyl rubber stopper, aluminum crimp seal |
|---|---|
| Nitrogen atmosphere | Headspace purged with dry nitrogen to retard methionine oxidation |
| Light sensitivity | Protect from direct light; store in original vial or amber container after reconstitution |
| Reconstitution solvent | Bacteriostatic water for injection (multi-dose use) or sterile water for injection (single-use); swirl gently, do not vortex; avoid agitation that promotes Met oxidation |
| Recommended concentration | 0.5–2.0 mg/mL in reconstitution solvent |
| Freeze-thaw cycles | Maximum 3 cycles; aliquot working volumes prior to initial freeze to minimize repeated cycling |
| USP references | USP <659> Packaging and Storage Requirements; USP <1> Injections and Implanted Drug Products (context only) |
6.0 Analytical Methods
Lot release testing comprises the following determinations. Full chromatographic method parameters are described in Nexphoria Method Note NX-MN-002.
| Test | Method | Acceptance Criterion |
|---|---|---|
| Purity | RP-HPLC, C18 (4.6×250 mm, 5 µm), UV 220 nm; 0.1% TFA gradient in water/acetonitrile | ≥98% (area percent, main peak); Met(O) impurity reported separately |
| Identity | ESI-MS, positive mode; theoretical [M+H]⁺ = 964.4 Da (for 8-mer) | Observed mass within ±0.5 Da of theoretical; charge state envelope consistent with sequence |
| Appearance | Visual inspection under daylight-equivalent illumination (≥2000 lux) | White to off-white lyophilized powder; free of visible foreign matter |
| Moisture (Karl Fischer) | Coulometric KF titration, USP <921> | ≤6.0% w/w |
| Endotoxin | LAL kinetic-chromogenic, USP <85> | ≤0.5 EU/mg |
| Peptide content (AAA) | Amino acid analysis, acid hydrolysis at 110°C / 24 h, HPLC quantitation | Consistent with theoretical composition ±10% per residue |
ESI-MS is required for each production lot. Charge states of +2 and +3 are typically observed for TB-500 under standard electrospray conditions. Deconvoluted average mass is compared against the theoretical value for the acetylated octapeptide [5, 6].
7.0 References
- [1] Low T.L., Hu S.K., Goldstein A.L. (1981). Complete amino acid sequence of bovine thymosin beta 4: a thymic hormone that induces terminal deoxynucleotidyl transferase activity in thymocyte populations. Proceedings of the National Academy of Sciences, 78(2):1162–1166. DOI:10.1073/pnas.78.2.1162
- [2] Huff T., Müller C.S., Otto A.M., Netzker R., Hannappel E. (2001). β-thymosins, small acidic peptides with multiple functions. International Journal of Biochemistry & Cell Biology, 33(3):205–220. DOI:10.1016/S1357-2725(00)00087-X
- [3] Brot N., Weissbach H. (1983). Biochemistry and physiological role of methionine sulfoxide residues in proteins. Archives of Biochemistry and Biophysics, 223(1):271–281. DOI:10.1016/0003-9861(83)90592-1
- [4] Manning M.C., Chou D.K., Murphy B.M., Payne R.W., Katayama D.S. (2010). Stability of protein pharmaceuticals: an update. Pharmaceutical Research, 27(4):544–575. DOI:10.1007/s11095-009-0045-6
- [5] Fenn J.B., Mann M., Meng C.K., Wong S.F., Whitehouse C.M. (1989). Electrospray ionization for mass spectrometry of large biomolecules. Science, 246(4926):64–71. DOI:10.1126/science.2675315
- [6] Roepstorff P., Fohlman J. (1984). Proposal for a common nomenclature for sequence ions in mass spectra of peptides. Biomedical Mass Spectrometry, 11(11):601. DOI:10.1002/bms.1200111109
- [7] Sewald N., Jakubik H.D. (2009). Peptides: Chemistry and Biology, 2nd ed. Wiley-VCH, Weinheim. ISBN: 978-3-527-31522-2. (Fmoc-SPPS methodology)
- [8] United States Pharmacopeia (2024). USP <659> Packaging and Storage Requirements; USP <85> Bacterial Endotoxins Test; USP <921> Water Determination. In USP–NF. United States Pharmacopeial Convention.