Effect of tween 20 on freeze-thawing- and agitation-induced aggregation of recombinant human factor XIII

Abstract

Agitation- and freeze-thawing-induced aggregation of recombinant human factor XIII (rFXIII) is due to interfacial adsorption and denaturation at the air-liquid and ice-liquid interfaces. The aggregation pathway proceeds through soluble aggregates to formation of insoluble aggregates regardless of the denaturing stimuli. A nonionic surfactant, polyoxyethylene sorbitan monolaurate (Tween 20), greatly reduces the rate of formation of insoluble aggregates as a function of surfactant concentration, thereby stabilizing native rFXIII. Maximum protection occurs at concentrations close to the critical micelle concentration (cmc), independent of initial protein concentration. To study the mechanistic aspects of the surfactant-induced stabilization, a series of spectroscopic studies were conducted. Electron paramagnetic resonance spectroscopy indicates that binding is not occurring between Tween 20 and either the native state or a folding intermediate state of rFXIII. Further, circular dichroism spectroscopy suggests that Tween 20 does not prevent the secondary structural changes induced upon guanidinium hydrochloride-induced unfolding. Taken together, these results imply that Tween 20 protects rFXIII against freeze-thawing- and agitation-induced aggregation primarily by competing with stress-induced soluble aggregates for interfaces, inhibiting subsequent transition to insoluble aggregates.

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 Lyoprotectant on the Solubility and Structure of Silk Sericin

“…Removing solvent, mostly water, is important because a liquid state protein or peptide is unstable to store. It is due to the hydrolysis or oxidation of protein in the presence of water, which results in various kinds of denaturation such as gelation, aggregation and degradation (Kreilgaard et al, 1998;Gabellieri et al, 2003;Gabellieri et al, 2006;Strambini et al, 2007). However, the removal of water may cause structural changes in the secondary and/or tertiary structure of protein.…”

<i>In Vitro</i> Culture of BMSCs on VEGF-SF-CS Three-Dimensional Scaffolds for Bone Tissue Engineering

“…In lyophilization course, usage of cryoprotector may reduce or even prevent influence of stress on activity of protein 57) . Silk protein and chitsan can be used as cryoprotector to well maintain activity of VEGF 58,59) . Alexander proved that conformation of protein treated with low concentration methanoll was basically not changed and covalent structure of protein molecule was not destroyed 60) .…”