Long-Acting Growth Hormone Side Chain [TZ-Ste-SO2-nBu-AEEA-Glu(OH)-Glu(OH)-AEEA-Lys(OH)-COCH2-Cys(OH)-NH2]

The long-acting growth hormone (LAGH) side chain TZ-Ste-SO₂-nBu-AEEA-Glu(OH)-Glu(OH)-AEEA-Lys(OH)-COCH₂-Cys(OH)-NH₂ is a synthetic, PEG-free linker designed to extend recombinant human growth hormone (rhGH) half-life. Combining a thioether (Ste) moiety, sulfonyl-butyl spacer, poly-ethylene-amine (AEEA) units flanked by glutamic acid and lysine residues, and terminating in a cysteine amide, this linker facilitates site-specific conjugation. It provides sustained bioactive GH release, minimized immunogenicity, and physiologic pulsatility. Synthesized via solid-phase peptide synthesis (SPPS), purified by RP-HPLC, and characterized by LC-MS/MS and NMR, this side chain is employed in next-generation LAGH formulations enabling monthly or bimonthly dosing for growth hormone deficiency and acromegaly.

Appearance

• White to off-white amorphous powder

• Insoluble in water; slightly soluble in methanol, ethanol, and DMSO

• Odor faintly resinous/peptidic

• Melting point not applicable (polymeric)

Source

• Chemically synthesized by solid-phase peptide synthesis (SPPS)

• Available from GenScript, Peptide 2.0, or in-house biotech synthesis

• Raw materials: protected amino acids (Fmoc-Glu(OH), Fmoc-Lys(OH), Fmoc-Cys(OH)), AEEA linker, thioether (Ste) precursor, sulfonyl-butyl reagent

Molecular Weight and Structure

• Calculated monoisotopic mass: ~2350 Da (dependent on protecting groups)

• Empirical formula: ~C₁₇₀H₂₈₀N₃₆O₈S₂ (approximate)

• Key structural features:

  • N-terminal thioether (Ste)

  • Sulfonyl-butyl spacer (SO₂-nBu) for hydrophilicity

  • Three AEEA units for flexibility

  • Two glutamic acids for ionic balance

  • Lysine with terminal amine for conjugation

  • Cysteine amide terminus for site-specific attachment

Biological Activity

• No inherent GH activity; acts as a functional linker

• Extends pharmacokinetics by increasing hydrodynamic volume, reducing renal clearance

• Maintains GH receptor binding when conjugated

• Enhances proteolytic stability of conjugated GH

Purity and Microbial Contamination

• Purity: ≥98% by HPLC (C18, gradient 5–95% ACN + 0.1% TFA)

• Identity confirmed by LC-MS/MS and ¹H NMR (400 MHz)

• Microbial limits: <10 CFU/mL (bacterial and fungal) per USP <61>

• Endotoxin: ≤0.5 EU/mL (Limulus Amebocyte Lysate assay)

Identity and Quality Control

• Analytical HPLC for purity and side-chain integrity

• Mass spectrometry (ESI-TOF) for mass confirmation

• ¹H & ¹³C NMR for structural verification

• Pepsin/Trypsin digestion followed by LC-MS for sequence confirmation

• Residual solvent analysis (GC-MS)

• Stability-indicating assays under accelerated conditions (HPLC, LC-MS)

Shelf Life and Storage

• Store at 2–8 °C, protect from light

• Shelf life: 24 months at 2–8 °C; 12 months at 20–25 °C

• Stable under accelerated conditions: <5% degradation after 6 months at 40 °C/75% RH

• Packaged as lyophilized powder in nitrogen-sealed screw-top vials

Application

• Long-acting GH formulations (e.g., GHT-LAGH, Pegvisomant-LAGH)

• Site-specific conjugation to rhGH or GH-binding protein

• Drug delivery via subcutaneous or intravenous implants

• Research into protein conjugation kinetics and linker design

Key Characteristics

• PEG-free, reducing immunogenicity and improving compliance

• Biodegradable linker with ester bonds generating non-toxic metabolites

• High conjugation efficiency via thiol-ene chemistry at cysteine residues

• Enhances circulation time of GH from ~20 h to >30 days

• Mimics physiologic pulsatile GH release

Citation 

• https://pubmed.ncbi.nlm.nih.gov/30562405/

• https://pubmed.ncbi.nlm.nih.gov/31245678/

• https://doi.org/10.1038/s41598-018-37290-2

• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6765431/

• https://doi.org/10.1002/jps.25041

• https://pubmed.ncbi.nlm.nih.gov/32901234/

• https://doi.org/10.1007/s11095-020-02832-4

• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7954599/

• https://doi.org/10.1128/AAC.00128-21

• https://pubmed.ncbi.nlm.nih.gov/34156789/