Tween protects recombinant human growth hormone against agitation-induced damage via hydrophobic interactions.
In the absence of surfactants, recombinant human growth hormone (rhGH) rapidly forms insoluble aggregates during agitation. The nonionic surfactant Tween 20, when present at Tween:protein molar ratios >4, effectively inhibits this aggregation. Differential scanning calorimetry (DSC) of rhGH solutions showed melting transitions that decreased by ca. 2 degrees C in the presence of Tween. Circular dichroism (CD) studies of the same thermal transition showed that the decrease is specific to the relatively high protein concentrations required for DSC. CD studies showed melting transitions that decreased 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 Tween with rhGH is characterized by a weak enthalpy of binding. For comparison, interferon-g, another protein which has been shown to bind Tween, also shows weak enthalpy of binding. Fluorescent probe binding studies and infrared spectroscopic investigations of rhGH secondary structure support suggestions in the literature (Bam, N. B.; Cleland, J. L., Randolph, T. W. Molten globule intermediate of recombinant human growth hormone: stabilization with surfactants. Biotechnol. Prog. 1996. 12, 801-809) that Tween binding is driven by hydrophobic interactions, with little perturbation of protein secondary structure.
Bam
N B
NB
SmithKline Beecham, King of Prussia, Pennsylvania 19406, USA.
Cleland
J L
JL
Yang
J
J
Manning
M C
MC
Carpenter
J F
JF
Kelley
R F
RF
Randolph
T W
TW
D002151:Calorimetry; D004347:Drug Interactions; D004355:Drug Stability; D019382:Human Growth Hormone / Q000737:chemistry*; D006801:Humans; D066298:In Vitro Techniques; D007372:Interferons / Q000737:chemistry; D011136:Polysorbates / Q000494:pharmacology*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013055:Spectrophotometry, Infrared; D013501:Surface-Active Agents / Q000378:metabolism*
D011136:Polysorbates; D011994:Recombinant Proteins; D013501:Surface-Active Agents; D019382:Human Growth Hormone; D007372:Interferons