Molten Globule Intermediate of Recombinant Human Growth Hormone: Stabilization with Surfactants

Abstract

We demonstrate that a surfactant-stabilized molten globule intermediate exists for recombinant human growth hormone (rhGH), is very hydrophobic, and tends to form aggregates. Characterization of this intermediate included equilibrium denaturation measured by electron paramagnetic resonance (EPR) and CD spectroscopy, assessment of aggregation during refolding, and fluorescence studies of its binding to the hydrophobic probe, 1-anilinonapthalene-8-sulfonate (1,8-ANS). We have found that at 4.5 M guanidinium hydrochloride (GuHCl), a molten globule intermediate of rhGH is stabilized and results in significant aggregation upon refolding. This intermediate is populated by the addition of the nonionic surfactant, Tween. This surfactant also reduces the extent of aggregation during refolding of rhGH from 4.5 M GuHCl. Overall, our studies reveal that rhGH forms a molten globule-like intermediate during folding and this intermediate self-associates. This self-association is reduced upon formation of a Tween-rhGH complex. Tween also binds to the native protein. Thus, nonionic surfactants such as Tween may act like molecular chaperones in facilitating protein folding while not altering the native conformation.

Lyophilization‐induced protein denaturation in phosphate buffer systems: Monomeric and tetrameric β‐galactosidase

“…During the freezing step of lyophilization, Tween may inhibit aggregation, possibly by competing for denaturing interfaces (i.e., ice interface or air±water interface) or by binding to protein states (native and folding intermediates). 39,40 Tween usually does not appear to inhibit protein unfolding during the drying step of lyophilization, but can facilitate refolding during rehydration.…”

Effect of Polysorbate 80 Concentration on Thermal and Photostability of a Monoclonal Antibody

“…Several mechanisms for protein stabilization by surfactants have been reported in the literature. Nonionic surfactants can protect proteins against surface-induced aggregation by competing with proteins for adsorption sites on surfaces, by binding to hydrophobic regions on the protein surface and thereby decreasing intermolecular interactions (8)(9)(10)(11), by increasing the free energy of protein unfolding (12), and finally, nonionic surfactants may act as chemical chaperone, favoring refolding over aggregation by binding transiently with partially folded protein molecules and sterically hindering intermolecular interactions that result in aggregation (13,14). Surfactants can also modulate adsorption loss and aggregation by coating interfaces and/or participating in protein-surfactant associations as demonstrated by Lee et al (15).…”

Effect of Polysorbate 80 Quality on Photostability of a Monoclonal Antibody

“…Polysorbates are widely used to protect biological drug products from unfolding, aggregation, and precipitation during shipping and handling (5)(6)(7). Polysorbates to be used in biotechnology products are required to be produced using strictly plant sources.…”