Tween protects recombinant human growth hormone against agitation-induced damage via hydrophobic interactions

In the absence of surfactants, recombinant human growth hormone (rhGH) rapi dly forms insoluble aggregates during agitation. The nonionic surfactant Tw een 20, when present at Tween:protein molar ratios > 4, effectively inhibit s this aggregation. Differential scanning calorimetry (DSC) of rhGH solutio ns showed melting transitions that decreased by ca. 2 degrees C in the pres ence of Tween. Circular dichroism (CD) studies of the same thermal transiti on showed that the decrease is specific to the relatively high protein conc entrations required for DSC. CD studies showed melting transitions that dec reased with lower protein concentrations. Tween has an insignificant effect on the melting transition of rhGH at lower protein concentrations (0.18 mg /mL). Injection titration microcalorimetry showed that the interaction of T ween with rhGH is characterized by a weak enthalpy of binding. For comparis on, interferon-g, another protein which has been shown to bind Tween, also shows weak enthalpy of binding. Fluorescent probe binding studies and infra red spectroscopic investigations of rhGH secondary structure support sugges tions in the literature (Bam, N. B.; Cleland, J. L., Randolph, T. W. Molten globule intermediate of recombinant human growth hormone: stabilization wi th surfactants. Biotechnol. Frog. 1996. 12, 801-809) that Tween binding is driven by hydrophobic interactions, with little perturbation of protein sec ondary structure.