title: Effects of Tween 20 and Tween 80 on the stability of Albutropin during agitation.
abstract: The objectives of this work were to determine the effects of nonionic surfactants (Tween 20 and Tween 80) on agitation-induced aggregation of the recombinant fusion protein, Albutropintrade mark (human growth hormone genetically fused to human albumin), and to characterize the binding interactions between the surfactants and the protein. Knowing the binding stoichiometry would allow a rational choice of surfactant concentration to protect the protein from surface-induced aggregation. Fluorescence spectroscopy and isothermal titration calorimetry (ITC) were employed to study Albutropin surfactant binding. Albutropin was agitated at 25 +/- 2 degrees C to induce aggregation, and samples were taken during a 96-h incubation. Size-exclusion chromatography (SEC-HPLC) (HPLC, high-performance liquid chromatography) was used to detect and quantify the extent of protein aggregation. The effect of surfactants on the protein's free energy of unfolding was determined using guanidine HCl as a denaturant. Tween 20 and Tween 80 had saturable binding to Albutropin with a molar binding stoichiometry of 10:1 and 9:1 (surfactant:protein), respectively. Binding of the surfactants to Albutropin increased the free energy of unfolding by over 1 and 0.6 kcal/mol, respectively. In protein samples that were agitated in the absence of surfactant, soluble aggregates were detected within 24 h, and there was almost complete loss of monomer to soluble aggregates by the end of the 96-h experiment. At the molar binding stoichiometry, Tween 20 and Tween 80 prevented the formation of soluble aggregates, even though the concentrations of surfactants were well below their critical micelle concentrations (CMC). Tween 20 and Tween 80 protected Albutropin against agitation-induced aggregation, even at concentrations below the CMC. Equilibrium unfolding data indicate that Tween confer protection by increasing the free energy of unfolding of Albutropin.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, Center for Pharmaceutical Biotechnology, University of Colorado Health Sciences Center, Denver, CO 80262, USA.
authors.forename: Danny K
authors.initials: DK
authors.lastname: Chou
authors.forename: Rajesh
authors.initials: R
authors.lastname: Krishnamurthy
authors.forename: Theodore W
authors.initials: TW
authors.lastname: Randolph
authors.forename: John F
authors.initials: JF
authors.lastname: Carpenter
authors.forename: Mark Cornell
authors.initials: MC
authors.lastname: Manning
mesh_terms: D004355:Drug Stability; D005453:Fluorescence; D019382:Human Growth Hormone / Q000737:chemistry*; D006801:Humans; D011136:Polysorbates / Q000494:pharmacology*; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D012709:Serum Albumin / Q000737:chemistry*
chemical_list: D011136:Polysorbates; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; D019382:Human Growth Hormone